UNA AMIDITES AND USES THEREOF

AMIDITES D'UNA ET LEURS UTILISATIONS

English Abstract

Disclosed herein are compositions and pharmaceutical formulations that comprise a binding moiety conjugated to a modified polynucleic acid molecule and a polymer. Also described herein include methods for treating a disease which utilize a composition or a pharmaceutical formulation comprising a binding moiety conjugated to a polynucleic acid molecule and a polymer.

French Abstract

L'invention concerne des compositions et des formulations pharmaceutiques qui comprennent un fragment de liaison conjugué à une molécule d'acide polynucléique modifié et un polymère. L'invention concerne également des méthodes pour traiter un cancer qui font intervenir une composition ou une formulation pharmaceutique comprenant un fragment de liaison conjugué à une molécule d'acide polynucléique et un polymère.

Claims

CLAIMS
WHAT IS CLAIMED IS:
1. A compound, or a pharmaceutically acceptable salt or solvate thereof,
having the
structure of Formula (II):
<IMG>
wherein:
<IMG>
Ring G is
X is 0, NH, or C(=0);
R1 is hydrogen, deuterium, halogen, substituted or unsubstituted C1-C10 alkyl,
substituted or
unsubstituted C2-Cio alkenyl, substituted or unsubstituted C2-Cio alkynyl,
substituted or
unsubstituted fluoroalkyl, substituted or unsubstituted
heteroalkyl,
substituted or unsubstituted cycloalkyl, substituted or unsubstituted
heterocycloalkyl,
substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl,
substituted or
unsubstituted -0-aryl, -0R6, -C(=0)R6, -C(=0)0R6, -C(=0)NR7R7, -OCH2C(=0)R6, -
OCH2C(=0)0R6, or-OCH2C(=0)NR7R7;
each R2 is independently hydrogen, deuterium, or substituted or unsubstituted
Ci-Cio alkyl;
or two R2 are taken together with the nitrogen atom to which they are attached
to form a
substituted or unsubstituted -C10 heterocycloalkyl;
R3 is hydrogen, 4,4'-dimethoxytrityl (DMT), -R6, -0R6, or -NR7R7;
each R4 is independently hydrogen, substituted or unsubstituted Ci-C6alkyl,
substituted or
unsubstituted aryl, or substituted or unsubstituted benzoyl;
R5 is -C(=0)R6;
each R6 and R7 are independently hydrogen, substituted or unsubstituted Ci-
C6alkyl, substituted
or unsubstituted Ci-C6fluoroalkyl, substituted or unsubstituted C3-
C6cycloalkyl,
substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted
aryl, or
substituted or unsubstituted heteroaryl.
-135-

2. The compound of claim 1, or a pharmaceutically acceptable salt or
solvate, thereof,
wherein: Rl is halogen, substituted or unsubstituted C1-C10 alkyl, substituted
or unsubstituted
Ci-Cio fluoroalkyl, substituted or unsubstituted Ci-C20 heteroalkyl,
substituted or unsubstituted
aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted
cycloalkyl, or
substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted -0-
aryl, -OH, -0R6, -
C(=0)R6, -C(=0)0R6, -C(=0)NR7R7, -OCH2C(=0)R6, or -OCH2C(=0)0R6, or -
OCH2C(=0)NR7R7.
3. The compound of claim 1 or 2, or a pharmaceutically acceptable salt or
solvate, thereof,
wherein: Rl is halogen, substituted or unsubstituted C1-C10 alkyl, substituted
or unsubstituted
C1-C20 heteroalkyl, substituted or unsubstituted -0-aryl, -OCH2C(=0)R6, or -
OCH2C(=0)0R6,
or -OCH2C(=0)NR7R7.
4. The compound of any one of claims 1-3, or a pharmaceutically acceptable
salt or solvate,
thereof, wherein: Rl is halogen or substituted or unsubstituted C1-C20
heteroalkyl.
5. The compound of any one of claims 1-4, or a pharmaceutically acceptable
salt or solvate,
<IMG>
thereof, wherein: Rl is
n is 0, 1, 2, 3, 4, or 5; and
le is hydrogen or substituted or unsubstituted C1-C3 alkyl.
6. The compound of claim 5, or a pharmaceutically acceptable salt or
solvate, thereof,
wherein: le is substituted or unsubstituted C1-C3 alkyl.
7. The compound of claim 5 or 6, or a pharmaceutically acceptable salt or
solvate, thereof,
wherein: le is methyl or ethyl.
8. The compound of any one of claims 5-7, or a pharmaceutically acceptable
salt or solvate,
thereof, wherein n is 1 or 2.
9. The compound of any one of claims 1-3, or a pharmaceutically acceptable
salt or solvate,
thereof, wherein: Rl is -OCH2C(=0)NR7R7; wherein each R7 is independently
hydrogen or
substituted or unsubstituted C1-C3 alkyl.
1 O. The compound of any one of claims 1-9, or a pharmaceutically
acceptable salt or solvate
thereof, wherein:
<IMG>
Ring G is ; wherein each R4 is
independently hydrogen, or substituted or unsubstituted benzoyl.
-1 36-

11. The compound of any one of claims 1-9, or a pharmaceutically acceptable
salt or solvate
thereof, wherein:
<IMG>
Ring G is ;
wherein each R4 is independently hydrogen
or substituted or unsubstituted benzoyl; and R5 is -C(=0)R6.
12. The compound of any one of claims 1-9 or 11, or a pharmaceutically
acceptable salt or
solvate thereof, wherein:
<IMG>
Ring G is ; and R5 is -C(=0)CH(CH3)2.
13. The compound of any one of claims 1-12, or a pharmaceutically
acceptable salt or
solvate thereof, wherein each R2 is independently hydrogen, deuterium, or
substituted or
unsubstituted Ci-Cio alkyl.
14. The compound of any one of claims 1-13, or a pharmaceutically
acceptable salt or
solvate thereof, wherein each R2 is independently substituted or unsubstituted
Ci-Cio alkyl.
15. The compound of any one of claims 1-14, or a pharmaceutically
acceptable salt or
solvate thereof, wherein each R2 is independently isopropyl.
16. The compound of any one of claims 1-12, or a pharmaceutically
acceptable salt or
solvate thereof, wherein two R2 are taken together with the nitrogen atom to
which they are
attached to form a substituted or unsubstituted C3-Cio heterocycloalkyl.
17. The compound of any one of claims 1-16, or a pharmaceutically
acceptable salt or
solvate thereof, wherein X is 0; and R3 is 4,4'-dimethoxytrityl (DMT).
18. A compound, or a pharmaceutically acceptable salt or solvate thereof,
having the
structure of Formula (lla):
<IMG>
wherein:
-137-

<IMG>
Ring G is
X is 0, NH, or C(=0);
n is 0, 1, 2, 3, or 4;
le is substituted or unsubstituted C1-C6 alkyl;
each R2 is independently hydrogen, deuterium, or substituted or unsubstituted
Ci-Cio alkyl;
or two R2 are taken together with the nitrogen atom to which they are attached
to form a
substituted or unsubstituted C3 -C o cycloalkyl;
R3 is hydrogen, 4,4'-dimethoxytrityl (DMT), -R6, -0R6, or -NR7R7.
each R4 is independently hydrogen, substituted or unsubstituted C1-C6alkyl,
substituted or
unsubstituted aryl, or substituted or unsubstituted benzoyl;
R5 is -C(=0)R6;
each R6 and R7 are independently hydrogen, substituted or unsubstituted C1-
C6alkyl, substituted
or unsubstituted Ci-C6fluoroalkyl, substituted or unsubstituted C3-
C6cycloalkyl,
substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted
aryl, or
substituted or unsubstituted heteroaryl.
19. The compound of claim 18, or a pharmaceutically acceptable salt or
solvate thereof,
wherein:
<IMG>
Ring G is ; wherein each R4 is
independently hydrogen, acetyl isobutyl or substituted or unsubstituted
benzoyl.
20. The compound of claim 18 or 19, or a pharmaceutically acceptable salt
or solvate
thereof, wherein:
<IMG>
Ring G is
21. The compound of claim 18, or a pharmaceutically acceptable salt or
solvate thereof,
wherein:
-138-

<IMG>
Ring G is each R4
is independently hydrogen or
substituted or unsubstituted benzyl; and R5 is -C(=0)R6.
22. The compound of claim 18 or 21, or a pharmaceutically acceptable salt
or solvate
thereof, wherein:
<IMG>
Ring G is ; and R5 is -C(=0)CH(CH3)2.
23. The compound of claim 22, or a pharmaceutically acceptable salt or
solvate thereof,
wherein; R6 is substituted or unsubstituted Ci-C6 alkyl.
24. The compound of any one of claims 18-23, or a pharmaceutically
acceptable salt or
solvate thereof, wherein le is methyl or ethyl.
25. The compound of any one of claims 18-24, or a pharmaceutically
acceptable salt or
solvate thereof, wherein n is 1 or 2.
26. The compound of any one of claims 18-25, or a pharmaceutically
acceptable salt or
solvate thereof, wherein n is 1.
27. The compound of any one of claims 18-26, or a pharmaceutically
acceptable salt or
solvate thereof, wherein each R2 is independently hydrogen, deuterium, or
substituted or
unsub stituted Ci-Cio alkyl.
28. The compound of any one of claims 18-27, or a pharmaceutically
acceptable salt or
solvate thereof, wherein each R2 is independently substituted or unsubstituted
Ci-Cio alkyl.
29. The compound of any one of claims 18-28, or a pharmaceutically
acceptable salt or
solvate thereof, wherein each R2 is independently isopropyl.
30. The compound of any one of claims 18-26, or a pharmaceutically
acceptable salt or
solvate thereof, wherein two R2 are taken together with the nitrogen atom to
which they are
attached to form a substituted or unsubstituted C3-Cio heterocycloalkyl.
31. The compound of any one of claims 18-30, or a pharmaceutically
acceptable salt or
solvate thereof, wherein R3 is 4,4'-dimethoxytrityl (DMT).
32. The compound of any one of claims 1-31, or a pharmaceutically
acceptable salt or
solvate thereof, selected from:
-139-

<IMG>
-140-

<IMG>
33. The compound of any one of claims 1-32, or a pharmaceutically
acceptable salt or
solvate thereof, wherein the compound is suitable for the synthesis of
oligonucleotides.
34. A compound suitable for the synthesis of oligonucleotides selected
from:
<IMG>
-141-

<IMG>
35. An oligonucleotide conjugate of Formula (I):
A-B
Formula (I);
wherein,
A is a binding moiety;
B is an oligonucleotide comprising a compound of, or a compound derived from,
Formula (II-1),
(II-2), or (II-3); and
<IMG>
wherein:
-142-

<IMG>
Ring G is
<IMG>
X is 0, NH, or C(=0);
Ll and L2 are each independently absent or a linking moiety selected from a
phosphodiester, amide and ester linkage:
R1 is hydrogen, deuterium, halogen, substituted or unsubstituted C1-C10 alkyl,
substituted
or unsubstituted C2-C10 alkenyl, substituted or unsubstituted C2-Cio alkynyl,
substituted or unsubstituted Ci-C10 fluoroalkyl, substituted or unsubstituted
Cl-
C20 heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted
or
unsubstituted heteroaryl, substituted or unsubstituted -0-aryl, -0R6, -
C(=0)R6, -
C(=0)0R6, -C(=0)NR7R7, -OCH2C(=0)R6, -OCH2C(=0)0R6, or-
OCH2C(=0)NR7R7;
each R2 is independently hydrogen, deuterium, or substituted or unsubstituted
Ci-Cio
alkyl;
or two R2 are taken together with the nitrogen atom to which they are attached
to form a
substituted or unsubstituted C3 -C10 heterocycloalkyl;
R3 is hydrogen, 4,4'-dimethoxytrityl (DMT), -R6, -0R6, or -NR7R7;
each R4 is independently hydrogen, substituted or unsubstituted C1-C6alkyl,
substituted
or unsubstituted aryl, or substituted or unsubstituted benzoyl;
R5 is -C(=0)0R6; and
each R6 and R7 are independently hydrogen, substituted or unsubstituted C1-
C6alkyl,
substituted or unsubstituted C1-C6fluoroalkyl, substituted or unsubstituted C
3 -
C6cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or
unsubstituted aryl, or substituted or unsubstituted heteroaryl.
36. The oligonucleotide conjugate of claim 35, wherein B is an
oligonucleotide comprising a
compound of Formula (II-1).
37. The oligonucleotide conjugate of claim 35, wherein B is an
oligonucleotide comprising a
compound of Formula (II-2).
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38. The oligonucleotide conjugate of claim 35, wherein B is an
oligonucleotide comprising a
compound derived from Formula (II-3).
39. The oligonucleotide conjugate of any one of claims 35-38, wherein the
oligonucleotide is
an RNA oligonucleotide.
40. The oligonucleotide conjugate of any one of the claims 35-39,
comprising at least one
modification.
41. The oligonucleotide conjugate of any one of the claims 35-40,
comprising at least one 2'
modified nucleotide.
42. The oligonucleotide conjugate of any one of the claims 35-41,
comprising at least one 2'
modified nucleotide selected from 2'-0-methyl, 2'-0-methoxyethyl (2'-0-M0E),
2'-0-
aminopropyl, 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-0-aminopropyl (2'-0-AP), 2'-0-
dimethylaminoethyl (2'-0-DMA0E), 2'-0-dimethylaminopropyl (2'-0-DMAP), 2'-0-
dimethylaminoethyloxyethyl (2'-0-DMAEOE), or 2'-0-N-methylacetamido (2'-0-NMA)
modified nucleotide.
43. The oligonucleotide conjugate of any one of claims 35-42, comprising at
least one 2'
modified nucleotide selected from locked nucleic acid (LNA) or ethylene
nucleic acid (ENA).
44. The oligonucleotide conjugate of any one of the claims 35-43,
comprising at least one
modified internucleotide linkage.
45. The oligonucleotide conjugate of any one of the claims 35-43,
comprising at least three
modified internucleotide linkage.
46. The oligonucleotide conjugate of any one of the claims 35-44,
comprising at least one
modified internucleotide linkage selected from a phosphorothioate, a
phosphorodithioate, a
methylphosphonate, a phosphotriester, or an amide linkage.
47. The oligonucleotide conjugate of claim 35, wherein the compound of
Formula (II-2) is
located at the 3'-terminus of the oligonucleotide.
48. The oligonucleotide conjugate of claim 35, wherein the compound of
Formula (II-1) is
located at an internal position within the oligonucleotide.
49. The oligonucleotide conjugate of any one of the claims 35-48, wherein
Formula (I)
further comprises C to form A-B-C (Formula I-A), wherein A and C are not
attached to B at the
same terminus.
50. The oligonucleotide conjugate of claim 49, wherein:
the compound of Formula (II-2) is located at the 5'-terminus of the
oligonucleotide; and
the binding moiety is conjugated to the 3'-terminus of the oligonucleotide.
51. The oligonucleotide conjugate of claim 49, wherein:
the compound of Formula (II-2) is located at the 3'-terminus of the
oligonucleotide; and
-144-

the binding moiety is conjugated to the 5'-terminus of the oligonucleotide.
52. The oligonucleotide conjugate of claim 49, wherein:
the compound of Formula (II-1) is located in the interior of the of the
oligonucleotide;
and
the binding moiety is conjugated to the 5'-terminus or the 3'-terminus of the
oligonucleotide.
53. The oligonucleotide conjugate of any one of the claims 49-52, wherein
the binding
moiety comprises an antibody or a binding fragment thereof
54. The oligonucleotide conjugate of any one of the claims 49-53, wherein
the binding
moiety comprises a humanized antibody or binding fragment thereof, a chimeric
antibody or
binding fragment thereof, a monoclonal antibody or binding fragment thereof, a
monovalent
Fab', a divalent Fab2, a single-chain variable fragment (scFv), a diabody, a
minibody, a
nanobody, a single-domain antibody (sdAb), or a camelid antibody or binding
fragment thereof
55. The oligonucleotide conjugate of claim 49, wherein the binding moiety
comprises a
peptide or a small molecule.
56. The oligonucleotide conjugate of any one of the claims 35-55,
comprising from about 8
to about 50 nucleotides.
57. The oligonucleotide conjugate of any one of the claims 35-56,
comprising from about 10
to about 30 nucleotides.
58. The oligonucleotide conjugate of any one of the claims 35-57, wherein
B:
comprises an RNA oligonucleotide;
has a length from about 10 to about 30 nucleotides;
comprises at least one 2' modified nucleotide; and
comprises at least one modified internucleotide linkage.
59. The oligonucleotide conjugate of any one of the claims 35-58, wherein B
hybridizes to at
least 8 contiguous bases of a target gene sequence.
60. The oligonucleotide conjugate of any one of the claims 35-59, wherein B
mediates RNA
interference.
61. The oligonucleotide conjugate of any one of the claims 35-60, wherein B
is a sense
strand.
62. The oligonucleotide conjugate of claim 61, wherein B is hybridized with
a second
oligonucleotide to form a double-stranded oligonucleic acid molecule.
63. The oligonucleotide conjugate of claim 62, wherein the second
oligonucleotide is an
antisense strand.
-145-

64. The oligonucleotide conjugate of claim 62 or 63, wherein the second
oligonucleotide is
an RNA oligonucleotide.
65. The oligonucleotide conjugate of any one of the claims 58-64, wherein
the second
oligonucleotide comprises at least one modification.
66. The oligonucleotide conjugate of any one of the claims 58-65, wherein
the second
oligonucleotide comprises a compound of Formula (II) or a compound of Formula
(Ha).
67. The oligonucleotide conjugate of claim 66, wherein the second
oligonucleotide
comprises at least one 2' modified nucleotide.
68. The oligonucleotide conjugate of claim 66 or 67, wherein the second
oligonucleotide
comprises at least one 2' modified nucleotide selected from 2'-0-methyl, 2'-0-
methoxyethyl (2'-
0-M0E), 2'-0-aminopropyl, 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-0-aminopropyl (2'-0-
AP), 2'-0-
dimethylaminoethyl (2'-0-DMA0E), 2'-0-dimethylaminopropyl (2'-0-DMAP), 2'-0-
dimethylaminoethyloxyethyl (2'-0-DMAEOE), or 2'-0-N-methylacetamido (2'-0-NMA)
modified nucleotide.
69. The oligonucleotide conjugate of any one of the claims 66-68, wherein
the second
oligonucleotide comprises at least one 2' modified nucleotide selected from
locked nucleic acid
(LNA) or ethylene nucleic acid (ENA).
70. The oligonucleotide conjugate of any one of the claims 66-69, wherein
the second
oligonucleotide comprises at least one modified internucleotide linkage.
71. The oligonucleotide conjugate of any one of the claims 66-70, wherein
the second
oligonucleotide comprises at least one modified internucleotide linkage
selected from a
phosphorothioate linkage or a phosphorodithioate linkage.
72. The oligonucleotide conjugate of any claim 49, wherein C is a polymer.
73. The oligonucleotide conjugate of claim 72, wherein the polymer
comprises polyethylene
glycol.
74. The oligonucleotide conjugate of any one of the claims 65-73, wherein
the
oligonucleotide comprises a sense strand and an antisense strand:
wherein the sense strand is an RNA oligonucleotide;
is conjugated to a binding moiety, a polymer, or a combination thereof;
is from about 10 to about 30 nucleotides;
comprises at least one 2' modified nucleotide; and
comprises at least one modified internucleotide linkage
and wherein the antisense strand:
is an RNA oligonucleotide;
is from about 10 to about 30 nucleotides;
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comprises at least one 2' modified nucleotide; and
comprises at least one modified internucleotide linkage.
75. A method of treating a subject having a disease or condition
characterized with a
defective protein expression, comprising administering to the subject an
oligonucleotide
conjugate of claims 35-74 to modulate expression of a gene encoding the
protein, thereby
treating the disease or condition characterized with the defective protein
expression.
76. A method of treating a subject having a disease or condition
characterized with an
overexpressed protein expression, comprising administering to the subject an
oligonucleotide
conjugate of claims 35-74 to modulate expression of a gene encoding the
protein, thereby
treating the disease or condition characterized with the overexpressed protein
expression.
77. The method of claim 75 or 76, wherein the disease or condition is a
cancer.
78. The method of claim 75 or 76, wherein the disease or condition is a
neuromuscular
disease, a muscle dystrophy, a muscle atrophy, a muscle wasting, a genetic
disease, cancer, a
hereditary disease, or a cardiovascular disease.
79. The method of any one of the claims 75-78, wherein the subject is a
human.
80. A kit comprising a compound of claims 1-32 or an oligonucleotide
conjugate of claims
35-74.
-147-

Description

CA 03142337 2021-11-30
WO 2020/247818 PCT/US2020/036420
UNA AMIDITES AND USES THEREOF
CROSS-REFERENCE
[0001] This application claims benefit of U.S Provisional Patent Application
No. 62/858,289
filed June 6, 2019, which is incorporated herein by reference in its entirety.
SEQUENCE LISTING
[0001.1] The instant application contains a Sequence Listing which has been
submitted
electronically in ASCII format and is hereby incorporated by reference in its
entirety. Said
ASCII copy, created on June 5, 2020, is named 45532-735 601 SL.txt and is
3,172,165 bytes in
size.
BACKGROUND OF THE DISCLOSURE
[0002] Gene suppression by RNA-induced gene silencing provides several levels
of control:
transcription inactivation, small interfering RN.A (siRNA)-induced rn.RNA
degradation, and
siRNA-induced transcriptional attenuation. In some instances, RNA interference
(RNAi)
provides long lasting effect over multiple cell divisions. As such, RNAi
represents a viable
method useful for drug target validation, gene function analysis, pathway
analysis, and disease
therapeutics.
SUMMARY OF THE DISCLOSURE
[0003] Disclosed herein, in certain embodiments, is a compound according to
Formula (II):
XR3
NCCI'V(30
,
R2N R2
R1 45 Formula (II);
wherein,
R5
HN H
er0 NR4R4
nrNR4R4
Ns( NyNH
Ring G is 0 0 0 , or 7.4 =
X is 0, NH, or C(=0);
R1 is hydrogen, deuterium, halogen, substituted or unsubstituted Ci-Cio alkyl,
substituted or
unsubstituted C2-C10 alkenyl, substituted or unsubstituted C2-C10 alkynyl,
substituted or
unsubstituted C1-C10 fluoroalkyl, substituted or unsubstituted C1-
C20heteroalkyl,
substituted or unsubstituted cycloalkyl, substituted or unsubstituted
heterocycloalkyl,
-1-

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substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl,
substituted or
unsubstituted -0-aryl, -0R6, -C(=0)R6, -C(=0)0R6, -C(=0)NR7R7, -OCH2C(=0)R6, -
OCH2C(=0)0R6, or-OCH2C(=0)NR7R7;
each R2 is independently hydrogen, deuterium, or substituted or unsubstituted
Ci-Cio alkyl;
or two R2 are taken together with the nitrogen atom to which they are attached
to form a
substituted or unsubstituted C3-Cio heterocycloalkyl;
R3 is hydrogen, 4,4'-dimethoxytrityl (DMT), -R6, -0R6, or -NR7R7;
each R4 is independently hydrogen, substituted or unsubstituted C1-C6alkyl,
substituted or
unsubstituted aryl, or substituted or unsubstituted benzoyl;
R5 is -C(=0)R6;
each R6 and R7 are independently hydrogen, substituted or unsubstituted C1-
C6alkyl, substituted
or unsubstituted Ci-C6fluoroalkyl, substituted or unsubstituted C3-
C6cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or
substituted or unsubstituted
heteroaryl.
[0004] In some instances, R1 is halogen, substituted or unsubstituted Ci-Cio
alkyl, substituted
or unsubstituted Ci-Cio fluoroalkyl, substituted or unsubstituted Ci-C20
heteroalkyl, substituted
or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or
unsubstituted
cycloalkyl, or substituted or unsubstituted heterocycloalkyl, substituted or
unsubstituted -0-aryl,
-OH, -0R6, -C(=0)R6, -C(=0)0R6, -C(=0)NR7R7, -OCH2C(=0)R6, or -OCH2C(=0)0R6,
or -
OCH2C(=0)NR7R7. In some instances, R1 is halogen, substituted or unsubstituted
Ci-Cio alkyl,
substituted or unsubstituted Ci-Cm heteroalkyl, substituted or unsubstituted -
0-aryl, -
OCH2C(=0)R6, or -OCH2C(=0)0R6, or -OCH2C(=0)NR7R7. In some instances, R1 is
halogen
or substituted or unsubstituted Ci-Cm heteroalkyl.
,v0410). R8
[0005] In some instances, R1 is , where n is 0, 1, 2, 3, 4, or 5, and
le is
hydrogen or substituted or unsubstituted Ci-C3 alkyl. In some embodiments, le
is substituted or
unsubstituted Ci-C3 alkyl. In some embodiments, R8 is methyl or ethyl. In some
embodiments, n
is 1 or 2.
[0006] In some instances, R1 is -OCH2C(=0)NR7R7; wherein each R7 is
independently
hydrogen or substituted or unsubstituted Cl-C3 alkyl.
N R4R4
NvNyNH N N
[0007] In some instances, Ring G is 0 0 , or 0 , and
each R4 is independently hydrogen, or substituted or unsubstituted benzoyl. In
some instances,
-2-

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R5 R5
HN H HN H
o
\N \N
Ring G is \ , and R5
is -C(=0)CH(CH3)2. In some instances, Ring G is \
,---N
/y_Z-NR4R4
NeN..õ,1
or \ ,
wherein each R4 is independently hydrogen or substituted or unsubstituted
benzoyl; and R5 is -C(=0)R6.
[0008] In some embodiments, each R2 is independently hydrogen, deuterium, or
substituted or
unsubstituted Ci-Cio alkyl. In some embodiments, each R2 is independently
substituted or
unsubstituted Ci-Cio alkyl. In some embodiments, each R2 is independently
isopropyl. In some
embodiments, two R2 are taken together with the nitrogen atom to which they
are attached to
form a substituted or unsubstituted C3-Cio heterocycloalkyl. In some
embodiments, X is 0, and
R3 is 4,4'-dimethoxytrityl (DMT).
[0009] In certain embodiments, described herein is a compound according to
Formula (ha):
0 (:),(0)R5
NC P
R2 N,R2 xR3
(Formula Ha);
wherein:
R5
HN H
,--N
er0 r ).--NR4R4 ,NR4R4
..õ,(NyNH Ni(NyN
Ring G is 0 0 0 , or =
X is 0, NH, or C(=0);
n is 0, 1, 2, 3, or 4;
R8 is substituted or unsubstituted Ci-C6 alkyl;
each R2 is independently hydrogen, deuterium, or substituted or unsubstituted
Ci-Cio alkyl;
or two R2 are taken together with the nitrogen atom to which they are attached
to form a
substituted or unsubstituted C3 -Ciocycloalkyl;
R3 is hydrogen, 4,4'-dimethoxytrityl (DMT), -R6, -0R6, or -NR7R7.
-3-

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each R4 is independently hydrogen, substituted or unsubstituted C1-C6alkyl,
substituted or
unsubstituted aryl, or substituted or unsubstituted benzoyl;
R5 is -C(=0)R6;
each R6 and IC are independently hydrogen, substituted or unsubstituted C1-
C6alkyl, substituted
or unsubstituted C1-C6fluoroalkyl, substituted or unsubstituted C3-
C6cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or
substituted or unsubstituted
heteroaryl.
er0
e
nrNR4R4
N y NH Nic N y NH N N
y
[0010] In some instances, Ring G is 0 0 , or 0 , and
each R4 is independently hydrogen, acetyl isobutyl or substituted or
unsubstituted benzoyl. In
R5
Hr
NvN y NH
some instances, Ring G is 0 . In some instances,
Ring G is or
rN NR4R4
, and each R4 is independently hydrogen or substituted or unsubstituted
benzyl;
R5
HN H
\N
and R5 is -C(=0)R6. In some instances, Ring G is , and R5 is -
C(=0)CH(CH3)2.
[0011] In some instances, R6 is substituted or unsubstituted Ci-C6 alkyl.
Alternatively and/or
additionally, le is methyl or ethyl. Alternatively and/or additionally, n is 1
or 2. In some
embodiments, n is 1. In some embodiments, each R2 is independently hydrogen,
deuterium, or
substituted or unsubstituted Ci-Cio alkyl. In some embodiments, each R2 is
independently
substituted or unsubstituted Ci-Cio alkyl. In some embodiments, each R2 is
independently
isopropyl. In some embodiments, two R2 are taken together with the nitrogen
atom to which they
are attached to form a substituted or unsubstituted C3-Cio heterocycloalkyl.
In some instances,
R3 is 4,4'-dimethoxytrityl (DMT).
[0012] In some instances, the compound is selected from
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ODMT e'ro
ODMT
cNyNH
l\t,,, N NH 0
0 ()
1 0
0 0 P
0' H
0 I Nj H NC) C)
NC) (1) I
ODMT
ONTNH H el
0 ODMT nrN
? 0 1 0 OH
1:)yN N
Y
N
NC) (:)
rl Lo o
T
0'13N H
? NC) 0
,
ODMT ODMT
I\OcNyNH 1\0(NyNH
0 0
? Or
0,P,N 0 0
0,P,N
1 F
NC) HN NC)
,
ODMT (:) ODMT (:)
ih%.,0NyNH ih%.,0NyNH
0,P,N P
0 N
NC) NC)
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.......ZN
N
ODMT ODMT
OyNN 1%%,.ON..,.,.N
0 1 0 0 1 LO
O'll'N H 0'11)N H
NC) 0 NC) C) ,and
,
0
NDMT
1%=,0eN NH
0
9 1 OH
0 N2
NC) 0 .
[0013] In some embodiments, the compound is suitable for the synthesis of
oligonucleotides.
[0014] In certain embodiments, disclosed herein is an oligonucleotide
comprising a compound
of Formula (II) or Formula (ha).
[0015] Disclosed herein, in certain embodiments, are compositions and
pharmaceutical
formulations that comprise a binding moiety conjugated to a polynucleic acid
molecule and
optionally a polymer. Thus, disclosed herein includes an oligonucleotide
conjugate of Formula
(I): A-B, where A is a binding moiety, and B is an oligonucleotide comprising
a compound of,
)(1_2
Lo
or a compound derived from Formula (II-1), (II-2), or (II-3), R1 41)
(II- 1 ),
)(1_2
LJ 0 xR3
0õ0
0 NC P 0
1
(1)
(II-2), or R2 N` R2
R1 CI
(II-3), where Ring G is Ny NH
R1
µi(
0 ,
R5
,
N( N H
,-N
NR4R4 NR4R4
o
N N
,õ,(NyNH NyN H
N
0 0 , , or \ , X
is 0, NH, or C(=0), Ll
\
and L2 are each independently absent or a linking moiety selected from a
phosphodiester, amide
and ester linkage, R1 is hydrogen, deuterium, halogen, substituted or
unsubstituted Ci-Cio alkyl,
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substituted or unsubstituted C2-Cio alkenyl, substituted or unsubstituted C2-
Cio alkynyl,
substituted or unsubstituted Ci-Cio fluoroalkyl, substituted or unsubstituted
Ci-C20 heteroalkyl,
substituted or unsubstituted cycloalkyl, substituted or unsubstituted
heterocycloalkyl, substituted
or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or
unsubstituted -0-
aryl, -0R6, -C(=0)R6, -C(=0)0R6, -C(=0)NR7R7, -OCH2C(=0)R6, -OCH2C(=0)0R6, or-
OCH2C(=0)NR7R7, each R2 is independently hydrogen, deuterium, or substituted
or
unsubstituted Ci-Cio alkyl, or two R2 are taken together with the nitrogen
atom to which they are
attached to form a substituted or unsubstituted C3-Cio heterocycloalkyl, R3 is
hydrogen, 4,4'-
dimethoxytrityl (DMT), -R6, -0R6, or -NR7R7, each R4 is independently
hydrogen, substituted or
unsubstituted C1-C6alkyl, substituted or unsubstituted aryl, or substituted or
unsubstituted
benzoyl, R5 is -C(=0)0R6, and each R6 and R7 are independently hydrogen,
substituted or
unsubstituted C1-C6alkyl, substituted or unsubstituted Ci-C6fluoroalkyl,
substituted or
unsubstituted C3-C6cycloalkyl, substituted or unsubstituted heterocycloalkyl,
substituted or
unsubstituted aryl, or substituted or unsubstituted heteroaryl. In some
embodiments, a
compound derived from a formula (e.g., a derivative compound from Formula 11-
3) comprises a
modified form of the formula with a substituted group. For example, in one
embodiment, a
compound derived from Formula 11-3 has a phosphodiester group, phosphotriester
group or a
phosphorothioate group substituting phosphonamidite group.
[0016] In some embodiments, the oligonucleotide is an RNA oligonucleotide. In
some
embodiments, the oligonucleotide comprises at least one modification, or at
least one 2'
modified nucleotide, which is selected from 2'-0-methyl, 2'-0-methoxyethyl (2'-
0-M0E), 2'-0-
aminopropyl, 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-0-aminopropyl (2'-0-AP), 2'-0-
dimethylaminoethyl (2'-0-DMA0E), 2'-0-dimethylaminopropyl (2'-0-DMAP), 2'-0-
dimethylaminoethyloxyethyl (2'-0-DMAEOE), or 2'-0-N-methylacetamido (2'-0-NMA)
modified nucleotide, or alternatively selected from locked nucleic acid (LNA)
or ethylene
nucleic acid (ENA). Alternatively and/or additionally, the oligonucleotide
comprises at least one
modified internucleotide linkage, optionally selected from a phosphorothioate,
a
phosphorodithioate, a methylphosphonate, a phosphotriester, or an amide
linkage.
[0017] In some embodiments, the derivative compound of Formula (II-3) is
located at the 5'-
terminus of the oligonucleotide. Alternatively and/or additionally, the
compound of Formula (II-
2) is located at the 3'-terminus of the oligonucleotide. Alternatively and/or
additionally, the
derivative compound of Formula (II-3) is located at the 5'-terminus of the
oligonucleotide; and
the binding moiety is conjugated to the 3'-terminus of the oligonucleotide.
Alternatively and/or
additionally, the compound of Formula (II-1) is located at an internal
position within the
oligonucleotide. Alternatively and/or additionally, the compound of Formula
(II-2) is located at
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the 5'-terminus of the oligonucleotide; and the binding moiety is conjugated
to the 3'-terminus of
the oligonucleotide. Alternatively and/or additionally, the compound of
Formula (II-2) is located
at the 3'-terminus of the oligonucleotide, and the binding moiety is
conjugated to the 5'-terminus
of the oligonucleotide. Alternatively and/or additionally, the compound of
Formula (II-1) is
located in the interior of the of the oligonucleotide , and the binding moiety
is conjugated to the
5'-terminus or the 3'-terminus of the oligonucleotide. In some embodiments,
Formula (I) further
comprises C to form A-B-C (Formula I-A), wherein A and C are not attached to B
at the same
terminus. In some embodiments, C is a polymer, which can comprise polyethylene
glycol.
[0018] In some embodiments, the binding moiety comprises an antibody or a
binding
fragment thereof In some embodiments, the binding moiety comprises a humanized
antibody or
binding fragment thereof, a chimeric antibody or binding fragment thereof, a
monoclonal
antibody or binding fragment thereof, a monovalent Fab', a divalent Fab2, a
single-chain
variable fragment (scFv), a diabody, a minibody, a nanobody, a single-domain
antibody (sdAb),
or a camelid antibody or binding fragment thereof. In some embodiments, the
binding moiety
comprises a peptide or a small molecule.
[0019] In some embodiments, the oligonucleotide comprises from about 8 to
about 50
nucleotides, or from about 10 to about 30 nucleotides.
[0020] In some embodiments, B of Formula I comprises an RNA oligonucleotide,
has a length
from about 10 to about 30 nucleotides, comprises at least one 2' modified
nucleotide, and
comprises at least one modified internucleotide linkage. In some embodiments,
B hybridizes to
at least 8 contiguous bases of a target gene sequence. In some embodiments, B
mediates RNA
interference. In some embodiments, B is a sense strand. In some embodiments, B
is hybridized
with a second oligonucleotide to form a double-stranded oligonucleic acid
molecule. In some
embodiments, the second oligonucleotide is an antisense strand. In some
embodiments, the
second oligonucleotide is an RNA oligonucleotide. In some embodiments, the
second
oligonucleotide comprises at least one modification. In some embodiments, the
second
oligonucleotide comprises a compound of Formula (II) or a compound of Formula
(ha). In some
embodiments, the second oligonucleotide comprises at least one 2' modified
nucleotide. In some
embodiments, the second oligonucleotide comprises at least one 2' modified
nucleotide selected
from 2'-0-methyl, 2'-0-methoxyethyl (2'-0-M0E), 2'-0-aminopropyl, 2'-deoxy, 2'-
deoxy-2'-
fluor , 2'-0-aminopropyl (2'-0-AP), 2'-0-dimethylaminoethyl (2'-0-DMA0E), 2'-0-
dimethylaminopropyl (2'-0-DMAP), 2'-0- dimethylaminoethyloxyethyl (2'-0-
DMAEOE), or 2'-
0-N-methylacetamido (2'-0-NMA) modified nucleotide, or alternatively and/or
additionally
selected from locked nucleic acid (LNA) or ethylene nucleic acid (ENA).
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[0021] In some embodiments, the second oligonucleotide comprises at least one
modified
internucleotide linkage. In some embodiments, the second oligonucleotide
comprises at least
one, or at least three modified internucleotide linkage selected from a
phosphorothioate linkage
or a phosphorodithioate linkage.
[0022] In some embodiments, the oligonucleotide is a sense strand, is an RNA
oligonucleotide, is conjugated to a binding moiety, a polymer, or a
combination thereof, is from
about 10 to about 30 nucleotides, comprises at least one 2' modified
nucleotide, and comprises at
least one modified internucleotide linkage.
[0023] In some embodiments, also described herein include methods for treating
a disease or
condition (e.g., cancer) that utilize a composition or a pharmaceutical
formulation comprising a
binding moiety conjugated to a polynucleic acid molecule and optionally a
polymer. Thus,
disclosed herein includes a method of treating a subject having a disease or
condition
characterized with a defective protein expression, comprising administering to
the subject an
oligonucleotide conjugate described herein to modulate expression of a gene
encoding the
protein, thereby treating the disease or condition characterized with the
defective protein
expression. Also disclosed herein includes a method of treating a subject
having a disease or
condition characterized with an overexpressed protein expression, comprising
administering to
the subject an oligonucleotide conjugate as described herein to modulate
expression of a gene
encoding the protein, thereby treating the disease or condition characterized
with the
overexpressed protein expression. In some embodiments, the disease or
condition is a cancer, a
neuromuscular disease, a muscle dystrophy, a muscle atrophy, a muscle wasting,
a genetic
disease, a hereditary disease, or a cardiovascular disease.
[0024] Disclosed herein, in certain embodiments, is a method of inhibiting the
expression of a
target gene in a primary cell of a patient, comprising administering a
molecule described above
to the primary cell. In some embodiments, the method is an in vivo method. In
some
embodiments, the patient is a human.
[0025] Disclosed herein, in certain embodiments, is an immuno-oncology therapy
comprising
a molecule described above for the treatment of a disease or disorder in a
patient in need thereof
[0026] Disclosed herein, in certain embodiments, is a kit comprising a
molecule described
above.
BRIEF DECRIPTION OF THE DRAWINGS
[0027] The following drawings form part of the present specification and are
included to
further demonstrate certain aspects of the present disclosure. The disclosure
may be better
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understood by reference to one or more of these drawings in combination with
the detailed
description of specific embodiments presented herein.
[0028] FIG. 1A shows a cartoon representation of a polynucleotide duplex,
comprising a
compound of Formula (II) attached to the 5' end of the guide (antisense)
strand, and further
comprising a binding moiety A, which may be attached to the polynucleotide
directly or via a
linker, attached to the: 5' end of the passenger (sense) strand.
[0029] FIG. 1B shows a cartoon representation of a polynucleotide duplex,
comprising one or
more compounds of Formula (II) attached to the 5' end of the guide (antisense)
strand, and
inserted into the antisense strand, and further comprising a binding moiety A,
which may be
attached to the polynucleotide directly or via a linker, attached to the: 5'
end of the passenger
(sense) strand.
[0030] FIG 2 shows a cartoon representation of an exemplary compound of
Formula (II) in an
oligonucleotide.
[0031] FIG. 3 shows a graph of MSTN knockdown in SJCRH30 cells in vitro using
four
different types of UNA siRNAs.
[0032] FIG. 4 shows a graph of MSTN knockdown in Gastrocnemius tissue in vivo
using a-
CD71-UNA siRNA conjugates.
DETAILED DESCRIPTION
[0033] Nucleic acid (e.g., RNAi) therapy is a targeted therapy with high
selectivity and
specificity. However, in some instances, nucleic acid therapy is also hindered
by poor
intracellular uptake, limited blood stability and non-specific immune
stimulation. To address
these issues, various modifications of the nucleic acid composition are
explored, such as for
example, novel linkers for better stabilizing and/or lower toxicity,
optimization of binding
moiety for increased target specificity and/or target delivery, and nucleic
acid polymer
modifications for increased stability and/or reduced off-target effect.
[0034] In some embodiments, the arrangement or order of the different
components that
make-up the nucleic acid composition further effects intracellular uptake,
stability, toxicity,
efficacy, and/or non-specific immune stimulation. For example, if the nucleic
acid component
includes a binding moiety, a polymer, and a polynucleic acid molecule (or
polynucleotide), the
order or arrangement of the binding moiety, the polymer, and/or the
polynucleic acid molecule
(or polynucleotide) (e.g., binding moiety-polynucleic acid molecule-polymer,
binding moiety-
polymer-polynucleic acid molecule, or polymer-binding moiety-polynucleic acid
molecule)
further effects intracellular uptake, stability, toxicity, efficacy, and/or
non-specific immune
stimulation.
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[0035] In some embodiments, described herein include an oligonucleotide
conjugate whose
arrangement of the nucleic acid components effects intracellular uptake,
stability, toxicity,
efficacy, and/or non-specific immune stimulation. In some instances, the
oligonucleotide
conjugate comprises a binding moiety conjugated to a polynucleic acid molecule
and a polymer.
In some embodiments, the oligonucleotide conjugate comprises a compound
according to
Formula (II) or Formula (Ha):
XR3
0{0)R8
0
/0õ0
NC P
R- R-
R1 0
OR3
R2N1:22
(II) or (Ha).
[0036] In some embodiments, an oligonucleotide conjugate comprising a binding
moiety
conjugated to a polynucleic acid molecule and a polymer arranged as described
herein enhances
intracellular uptake, stability, and/or efficacy. In some instances, an
oligonucleotide conjugate
comprising a binding moiety conjugated to a polynucleic acid molecule and a
polymer arranged
as described herein reduces toxicity and/or non-specific immune stimulation.
In some cases, the
oligonucleotide conjugate comprises a compound according to Formula (II) or
Formula (Ha):
(1)
Xi7t3
0,0)R8
0
NC
/0Põ0
R-, N, , R-
R1
R2NLR2 OR3
(II) or (Ha).
[0037] In additional embodiments, described herein include a kit, which
comprises one or
more of the molecules or compositions described herein.
Therapeutic Molecule Platform
[0038] In some embodiments, an oligonucleotide conjugate (e.g., a therapeutic
oligonucleotide
conjugate) described herein comprises a binding moiety conjugated to a
polynucleic acid
molecule comprising one or more modified nucleotides and a polymer. In some
embodiments,
the oligonucleotide conjugate comprises a compound according to Formula (I) or
Formula (I-A):
A-B
Formula (I);
wherein,
A is a binding moiety; and
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B is an oligonucleotide comprising a compound of Formula (II) or (ha)
A-B-C
Formula (I-A);
wherein,
A is a binding moiety; and
B is an oligonucleotide comprising a compound of Formula (II) or (ha)
C is optionally a polymer.
[0039] In some embodiments, the oligonucleotide comprises a compound according
to
Formula (II):
xR3
NC
R2N'IR2
(1)
Ri Formula (II);
wherein,
R5
HN H
\.r0 RO 4R4 //µ
NR4R4
NvNirNH NvNyNH NvNyN
Ring G is ir 0 0 0 , or =
X is 0, NH, or C(=0);
R1 is hydrogen, deuterium, halogen, substituted or unsubstituted Ci-Cio alkyl,
substituted or
unsubstituted C2-C10 alkenyl, substituted or unsubstituted C2-C10 alkynyl,
substituted or
unsubstituted C1-C10 fluoroalkyl, substituted or unsubstituted C1-C20
heteroalkyl,
substituted or unsubstituted cycloalkyl, substituted or unsubstituted
heterocycloalkyl,
substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl,
substituted or
unsubstituted -0-aryl, -0R6, -C(=0)R6, -C(=0)0R6, -C(=0)NR7R7, -OCH2C(=0)R6, -
OCH2C(=0)0R6, or-OCH2C(=0)NR7R7;
each R2 is independently hydrogen, deuterium, or substituted or unsubstituted
Ci-Cio alkyl;
or two R2 are taken together with the nitrogen atom to which they are attached
to form a
substituted or unsubstituted C3-C10 heterocycloalkyl;
R3 is hydrogen, 4,4'-dimethoxytrityl (DMT), -R6, -0R6, or -NR7R7;
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each R4 is independently hydrogen, substituted or unsubstituted C1-C6alkyl,
substituted or
unsubstituted aryl, or substituted or unsubstituted benzoyl;
R5 is -C(=0)R6;
each R6 and R7 are independently hydrogen, substituted or unsubstituted C1-
C6alkyl, substituted
or unsubstituted C1-C6fluoroalkyl, substituted or unsubstituted C3-
C6cycloalkyl,
substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted
aryl, or
substituted or unsubstituted heteroaryl.
[0040] In some embodiments of Formula (II), R1 is halogen, substituted or
unsubstituted Ci-
Cio alkyl, substituted or unsubstituted Ci-Cio fluoroalkyl, substituted or
unsubstituted Ci-C20
heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted
heteroaryl, substituted
or unsubstituted cycloalkyl, or substituted or unsubstituted heterocycloalkyl,
substituted or
unsubstituted -0-aryl, -OH, -0R6, -C(=0)R6, -C(=0)0R6, -C(=0)NR7R7, -
OCH2C(=0)R6, or -
OCH2C(=0)0R6, or -OCH2C(=0)NR7R7.
[0041] In some embodiments of Formula (II), R1 is halogen, substituted or
unsubstituted Ci-
Cio alkyl, substituted or unsubstituted Ci-C20 heteroalkyl, substituted or
unsubstituted -0-aryl, -
OCH2C(=0)R6, or -OCH2C(=0)0R6, or -OCH2C(=0)NR7R7. In some embodiments of
Formula
(II), R1 is halogen or substituted or unsubstituted C i-C20 heteroalkyl. In
some embodiments of
Formula (II), R1 is fluoro, choro, or bromo. In some embodiments of Formula
(II), R1 is fluoro.
=,,v00).R8
[0042] In some embodiments of Formula (II), R1 is ; n is 0, 1, 2, 3, 4,
or 5;
and le is hydrogen or substituted or unsubstituted Ci-C3 alkyl. In some
embodiments, n is 1, 2,
or 3. In some embodiments, n is 1 or 2. In some embodiments, n is 1. In some
embodiments, n is
2. In some embodiments, n is 0.
R8
[0043] In some embodiments of Formula (II), R1 is n ;
and le is substituted or
unsubstituted Ci-C3 alkyl. In some embodiments, R8 is methyl or ethyl. In some
embodiments,
R8 is methyl. In some embodiments, le is ethyl. In some embodiments, le is H.
e(:)
ir NH ,,,(NyNH
[0044] In some embodiments of Formula (II), Ring G is 0 0 , or
nrN R4 R4
NcNyN
; wherein each R4 is independently hydrogen or substituted or unsubstituted
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e\rO
NyNH
benzoyl. In some embodiments of Formula (II), ring B is 0
. In some embodiments
iõ,(NyNH
of Formula (II), Ring G is 0 . In some embodiments of Formula (II),
Ring G is
nrNR4R4
NvNyN
R5
HN H
,---N
)--NR4R4
\1s1õ
100451 In some embodiments of Formula (II), Ring G is, ( or
=
wherein each R4 is independently hydrogen or substituted or unsubstituted
benzoyl; and R5 is -
R5
HN H
C(=0)R6. In some embodiments of Formula (II), Ring G is, ;
wherein R5 is -
R5
HN H
C(=0)R6. In some embodiments of Formula (II), Ring G is ;
wherein R5 is -
,---N
C(=0)CH(CH3)2. In some embodiments of Formula (II), Ring G is \ ;
wherein
each R4 is independently hydrogen or substituted or unsubstituted benzoyl.
100461 In some embodiments of Formula (II), each R2 is independently hydrogen,
deuterium,
or substituted or unsubstituted Ci-C10 alkyl. In some embodiments of Formula
(II), each R2 is
independently substituted or unsubstituted Ci-Cio alkyl. In some embodiments
of Formula (II),
each R2 is isopropyl.
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[0047] In some embodiments of Formula (II), two R2 are taken together with the
nitrogen
atom to which they are attached to form a substituted or unsubstituted C3-Cio
heterocycloalkyl.
[0048] In some embodiments of Formula (II), X is 0 and R3 is 4,4'-
dimethoxytrityl (DMT).
[0049] In some embodiments, the oligonucleotide comprises a compound according
to
Formula (ha):
0 0o)R5
0õ.)HNC P0
R2 N , R2 xR3
(Formula Ha);
wherein:
R5
HN H
N 0
N
fis -N)---NR4R4
r iF WWI
NicNyNH NyN
N.,(N
Ring G is 0 0 0 , or N. =
X is 0, NH, or C(=0);
n is 0, 1, 2, 3, or 4;
R8 is substituted or unsubstituted Ci-C6 alkyl;
each R2 is independently hydrogen, deuterium, or substituted or unsubstituted
Ci-Cio alkyl;
or two R2 are taken together with the nitrogen atom to which they are attached
to form a
substituted or unsubstituted c3 -c iocycloalkyl;
R3 is hydrogen, 4,4'-dimethoxytrityl (DMT), -R6, -0R6, or -NR7R7.
each R4 is independently hydrogen, substituted or unsubstituted C1-C6alkyl,
substituted or
unsubstituted aryl, or substituted or unsubstituted benzoyl;
R5 is -C(=0)R6;
each R6 and R7 are independently hydrogen, substituted or unsubstituted C1-
C6alkyl, substituted
or unsubstituted C1-C6fluoroalkyl, substituted or unsubstituted C3-
C6cycloalkyl,
substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted
aryl, or
substituted or unsubstituted heteroaryl.
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e\rO
o
NicNyNH
[0050] In some embodiments of Formula (ha), Ring G is 0 0 , or
nrNR4R4
NvNyN
0 ;
wherein each R4 is independently hydrogen or substituted or unsubstituted
er0
y NH
benzyl. In some embodiments of Formula (ha), Ring G is 0
. In some embodiments
=,,,,(NyNH
of Formula (II), Ring G is 0
. In some embodiments of Formula (ha), Ring G is
nrNR4R4
NvNyN
0
R5
HN H
NR4R4
[0051] In some embodiments of Formula (ha), Ring G is or "µ= =
wherein each R4 is independently hydrogen or substituted or unsubstituted
benzyl; and R5 is -
R5
HN H
C(=0)R6. In some embodiments of Formula (ha), Ring G is ;
wherein R5 is -
R5
HN H
C(=0)R6. In some embodiments of Formula (ha), Ring G is ;
wherein R5 is -
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CA 03142337 2021-11-30
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,-N
Z-NR4R4
N
,õ.N.,"
C(=0)CH(CH3)2. In some embodiments of Formula (Ha), Ring G is \ ;
wherein
each R4 is independently hydrogen or substituted or unsubstituted benzoyl.
[0052] In some embodiments of Formula (Ha), n is 0, 1, 2, 3, 4, or 5. In some
embodiments of
Formula (Ha), n is 0, 1, 2, 3, or 4. In some embodiments of Formula (Ha), n is
1 or 2. In some
embodiments of Formula (Ha), n is 0. In some embodiments of Formula (Ha), n is
1. In some
embodiments of Formula (Ha), n is 2.
[0053] In some embodiments of Formula (Ha), R8 is hydrogen or substituted or
unsubstituted
Ci-C3 alkyl. In some embodiments of Formula (Ha), R8 is substituted or
unsubstituted Ci-C3
alkyl. In some embodiments of Formula (Ha), R8 is methyl or ethyl. In some
embodiments of
Formula (Ha), le is methyl.
[0054] In some embodiments of Formula (Ha), each R2 is independently hydrogen,
deuterium,
or substituted or unsubstituted Ci-C10 alkyl. In some embodiments of Formula
(Ha), each R2 is
independently substituted or unsubstituted Ci-C10 alkyl. In some embodiments
of Formula (Ha),
each R2 is isopropyl.
[0055] In some embodiments of Formula (Ha), two R2 are taken together with the
nitrogen
atom to which they are attached to form a substituted or unsubstituted C3-Cio
heterocycloalkyl.
[0056] In some embodiments of Formula (Ha), R3 is 4,4'-dimethoxytrityl (DMT).
[0057] In some embodiments of Formula (II) or (Ha), the compound is selected
from the
group consisting of:
ODMT
ODMT ero LOyrµ11.(NH
ODMT 1%,C o
L,O(N1.(NH
,hNe'rI.rNH 0 1
0 Cr 1 OH N Cr 1 O C 0H
,P,
0 N
0 N NC) 0
NC) 0 I LO
I
,
H
ODMT nrN
ODMT e..ro
ODMT e'ro
L,.0(NI.rNH 1%ChNI.rNH
L 0 0 0
9 1 OH
,P, Cr 1 Or
0 N 0 N 0 N
NC) 0 NC) HN NC)
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H
\ZN
ODMT eo
ODMT eo
ODMT 0
1%0,..1%11.rNH 10yNyNH
0 L
9 F F 0 0
0 0
0-1pN2 H 0
NC) NC) NC) ()
0 y
ODMT NDMT er
Lx0,1µ1õ, LO(rAl.rNH
L 0
9 0 N OH H
0
NC) ,and NC)
[0058] In some embodiments, the oligonucleotide comprises at least about 1,
about 2, about 3,
about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11,
about 12, about 13,
about 14, about 15, about 16, about 17, about 18, about 19, about 20, about
21, about 22, about
23, about 24, about 25, about 30, or more compounds of Formula (II) (e.g.,
Formula Ha). In
some cases, the oligonucleotide comprises at least about 1, about 2, about 3,
about 4, about 5,
about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13,
about 14, about 15, or
more compounds of Formula (II) (e.g., Formula Ha). In some instances, the
compounds of
Formula (II) (e.g., Formula ha) are in tandem within the oligonucleotide. In
other instances, the
compounds Formula (II) (e.g., Formula ha) are interspersed within the
oligonucleotide, with
nucleotides modified by one or more additional modification described below.
[0059] In some instances, the oligonucleotide comprises at least one of: from
about 5% to
about 100% modification, from about 10% to about 100% modification, from about
20% to
about 100% modification, from about 30% to about 100% modification, from about
40% to
about 100% modification, from about 50% to about 100% modification, from about
60% to
about 100% modification, from about 70% to about 100% modification, from about
80% to
about 100% modification, and from about 90% to about 100% modification, in
which the
modification is a compound of Formula (II) (e.g., Formula Ha). For example,
where the
oligonucleotide has 20 nucleosides, the oligonucleotide haying about 60%
modification
comprises about 12 nucleosides substituted with 12 Formula (II) (e.g., Formula
ha) compounds.
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[0060] In some embodiments, an oligonucleotide conjugate is a molecule as
illustrated:
A-B-C
[0061] In some embodiments, an oligonucleotide conjugate is a molecule as
illustrated:
4.1's
A-C-B
[0062] The ,\ as illustrated above is for representation purposes only
and
encompasses a humanized antibody or binding fragment thereof, chimeric
antibody or binding
fragment thereof, anti-human antibody, anti-murine antibody (e.g., anti-mouse
antibody, anti-rat
antibody, etc.), monoclonal antibody or binding fragment thereof, monovalent
Fab', divalent
Fab2, single-chain variable fragment (scFv), diabody, minibody, nanobody,
single-domain
antibody (sdAb), or camelid antibody or binding fragment thereof
Additional Modifications
[0063] In some embodiments, the additional modifications include synthetic or
artificial
nucleotide analogues or bases comprising modifications at one or more of
ribose moiety,
phosphate moiety, nucleoside moiety, or a combination thereof
[0064] In some embodiments, a modification at a 2' hydroxyl group include 2'-
deoxy, 2-
deoxy-2'-fluoro, 2'-0-aminopropyl (2'-0-AP), 2'-0-dimethylaminoethyl (2'-0-
DMA0E), 2'-0-
dimethylaminopropyl (2'-0-DMAP), 2'-0- dimethylaminoethyloxyethyl (2'-0-
DMAEOE), or 2'-
0-N-methylacetamido (2'-0-NMA).
[0065] In some embodiments, a nucleotide analogue comprises a modified base
such as, but
not limited to, 5-propynyluridine, 5-propynylcytidine, 6- methyladenine, 6-
methylguanine, N, N,
-dimethyladenine, 2-propyladenine, 2-propylguanine, 2-aminoadenine, 1-
methylinosine, 3-
methyluridine, 5-methylcytidine, 5-methyluridine and other nucleotides having
a modification at
the 5 position, 5- (2- amino) propyl uridine, 5-halocytidine, 5-halouridine, 4-
acetylcytidine, 1-
methyladenosine, 2-methyladenosine, 3-methylcytidine, 6-methyluridine, 2-
methylguanosine,
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7-methylguanosine, 2, 2-dimethylguanosine, 5- methylaminoethyluridine, 5-
methyloxyuridine,
deazanucleotides (such as 7-deaza- adenosine, 6-azouridine, 6-azocytidine, or
6-azothymidine),
5-methyl-2-thiouridine, other thio bases (such as 2-thiouridine, 4-
thiouridine, and 2-
thiocytidine), dihydrouridine, pseudouridine, queuosine, archaeosine, naphthyl
and substituted
naphthyl groups, any 0-and N-alkylated purines and pyrimidines (such as N6-
methyladenosine,
5-methylcarbonylmethyluridine, uridine 5-oxyacetic acid, pyridine-4-one, or
pyridine-2-one),
phenyl and modified phenyl groups such as aminophenol or 2,4, 6-trimethoxy
benzene, modified
cytosines that act as G-clamp nucleotides, 8-substituted adenines and
guanines, 5-substituted
uracils and thymines, azapyrimidines, carboxyhydroxyalkyl nucleotides,
carboxyalkylaminoalkyi nucleotides, and alkylcarbonylalkylated nucleotides. 5'-
modified
nucleotides also include those nucleotides that are modified with respect to
the sugar moiety, as
well as 5'-modified nucleotides having sugars or analogs thereof that are not
ribosyl. For
example, the sugar moieties, in some cases are or are based on, mannoses,
arabinoses,
glucopyranoses, galactopyranoses, 4'-thioribose, and other sugars,
heterocycles, or carbocycles.
The term nucleotide also includes what are known in the art as universal
bases. By way of
example, universal bases include but are not limited to 3 -nitropyrrole, 5-
nitroindole, or
nebularine.
[0066] In some instances, the modification at the 2' hydroxyl group is a 2'-0-
aminopropyl
modification in which an extended amine group comprising a propyl linker binds
the amine
group to the 2' oxygen. In some instances, this modification neutralizes the
phosphate-derived
overall negative charge of the oligonucleotide molecule by introducing one
positive charge from
the amine group per sugar and thereby improves cellular uptake properties due
to its zwitterionic
properties.
[0067] In some instances, the 5'-modified nucleotide is further modified at
the 2' hydroxyl
group in a locked or bridged ribose modification (e.g., locked nucleic acid or
LNA) in which the
oxygen molecule bound at the 2' carbon is linked to the 4' carbon by a
methylene group, thus
forming a 2'-C,4'-C-oxy-methylene-linked bicyclic ribonucleotide monomer.
[0068] In some embodiments, the additional modification further comprises a
morpholino, a
peptide nucleic acid (PNA), a methylphosphonate nucleotide, a thiolphosphonate
nucleotide, a
2'-fluoro N3-P5'-phosphoramidite, or a 1', 5'- anhydrohexitol nucleic acid
(HNA). Morpholino
or phosphorodiamidate morpholino oligo (PMO) comprises synthetic molecules
whose structure
mimics natural nucleic acid structure but deviates from the normal sugar and
phosphate
structures. In some instances, the five member ribose ring is substituted with
a six member
morpholino ring containing four carbons, one nitrogen, and one oxygen. In some
cases, the
ribose monomers are linked by a phosphordiamidate group instead of a phosphate
group. In such
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cases, the backbone alterations remove all positive and negative charges
making morpholinos
neutral molecules capable of crossing cellular membranes without the aid of
cellular delivery
agents such as those used by charged oligonucleotides. A non-limiting example
of a 5'-
modified morpholino oligonucleotide is illustrated below.
DMT
0=P¨NMe2
0
L,(0)Base
Morpholino
R'
[0069] In some embodiments, a 5'- modified morpholino or PM0 described above
is a PM0
comprising a positive or cationic charge. In some instances, the PM0 is
PM0plus (Sarepta).
PM0plus refers to phosphorodiamidate morpholino oligomers comprising any
number of (1-
piperazino)phosphinyiideneoxy, 0.-(4-(omega -guanidino-alkanoyl))-.
piperazino)phosphinylideneoxy linkages (e.g., as such those described in PCT
Publication No.
W02008/036127. In some cases, the PM0 is a PM0 described in U.S. Patent No.
7943762.
[0070] In some embodiments, a morpholino or PM0 described above is a PMO-X
(Sarepta).
In some cases, PMO-X refers to phosphorodiamidate morpholino oligomers
comprising at least
one linkage or at least one of the disclosed terminal modifications, such as
those disclosed in
PCT Publication No. W02011/150408 and U.S. Publication No. 2012/0065169.
[0071] In some embodiments, a morpholino or PM0 described above is a PM0 as
described
in Table 5 of U.S. Publication No. 2014/0296321.
[0072] In some embodiments, peptide nucleic acid (PNA) does not contain sugar
ring or
phosphate linkage and the bases are attached and appropriately spaced by
oligoglycine-like
molecules, therefore, eliminating a backbone charge.
Yo 0
1\1*-"NNse">µ.
PNA
[0073] In some embodiments, one or more modifications described above occur at
the
internucleotide linkage. In some instances, modified internucleotide linkage
includes, but is not
limited to, phosphorothioates; phosphorodithioates; methylphosphonates; 5'-
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alkylenephosphonates; 5'-methylphosphonate; 3'-alkylene phosphonates;
borontrifluoridates;
borano phosphate esters and selenophosphates of 3'-5'linkage or 2'-5'linkage;
phosphotriesters;
thionoalkylphosphotriesters; hydrogen phosphonate linkages; alkyl
phosphonates;
alkylphosphonothioates; arylphosphonothioates; phosphoroselenoates;
phosphorodiselenoates;
phosphinates; phosphoramidates; 3'- alkylphosphoramidates;
aminoalkylphosphoramidates;
thionophosphoramidates; phosphoropiperazidates; phosphoroanilothioates;
phosphoroanilidates;
ketones; sulfones; sulfonamides; carbonates; carbamates; methylenehydrazos;
methylenedimethylhydrazos; formacetals; thioformacetals; oximes;
methyleneiminos;
methylenemethyliminos; thioamidates; linkages with riboacetyl groups;
aminoethyl glycine;
silyl or siloxane linkages; alkyl or cycloalkyl linkages with or without
heteroatoms of, for
example, 1 to 10 carbons that are saturated or unsaturated and/or substituted
and/or contain
heteroatoms; linkages with morpholino structures, amides, or polyamides
wherein the bases are
attached to the aza nitrogens of the backbone directly or indirectly; and
combinations thereof.
[0074] In some instances, the modification is a methyl or thiol modification
such as
methylphosphonate or thiolphosphonate modification. Exemplary thiolphosphonate
nucleotide
(left), phosphorodithioates (center) and methylphosphonate nucleotide (right)
are illustrated
below.
DMT DMT
DMT
b¨NcOrbase
-0¨P=S -S¨P=S Me¨P=0
(3( (3(
3 end 3' end 3' end
[0075] In some instances, a 5'-modified nucleotide includes, but is not
limited to,
phosphoramidites illustrated as:
DMT
b¨N(Orbase
HN1
-04=0
3' end
[0076] In some instances, the modified internucleotide linkage is a
phosphorodiamidate
linkage. A non-limiting example of a phosphorodiamidate linkage with a
morpholino system is
shown below.
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DMT
0=P-NMe2
0
b,Base
0 -R
3 End
[0077] In some instances, the modified internucleotide linkage is a
methylphosphonate
linkage. A non-limiting example of a methylphosphonate linkage is shown below.
DMT e=r0
µ0-,\(010,N0NH
/ II
____________________________________ t
Me-P=0
Ois
3' end
[0078] In some instances, the modified internucleotide linkage is an amide
linkage. A non-
limiting example of an amide linkage is shown below.
ee)
DMT
N NH
_________________________________________ Y
= 0
OR
ONH
0 'Ft
3' end
[0079] In some embodiments, one or more modifications comprise a modified
phosphate
backbone in which the modification generates a neutral or uncharged backbone.
In some
instances, the phosphate backbone is modified by alkylation to generate an
uncharged or neutral
phosphate backbone. As used herein, alkylation includes methylation,
ethylation, and
propylation. In some cases, an alkyl group, as used herein in the context of
alkylation, refers to
a linear or branched saturated hydrocarbon group containing from 1 to 6 carbon
atoms. In some
instances, exemplary alkyl groups include, but are not limited to, methyl,
ethyl, n-propyl,
isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n- pentyl, isopentyl,
neopentyl, hexyl,
isohexyl, 1, 1 -dimethylbutyl, 2,2-dimethylbutyl, 3.3- dimethylbutyl, and 2-
ethylbutyl groups.
In some cases, a modified phosphate is a phosphate group as described in U.S.
Patent No.
9481905.
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[0080] In some embodiments, additional modified phosphate backbones comprise
methylphosphonate, ethylphosphonate, methylthiophosphonate, or
methoxyphosphonate. In
some cases, the modified phosphate is methylphosphonate. In some cases, the
modified
phosphate is ethylphosphonate. In some cases, the modified phosphate is
methylthiophosphonate. In some cases, the modified phosphate is
methoxyphosphonate.
[0081] In some embodiments, one or more modifications further optionally
include
modifications of the ribose moiety, phosphate backbone and the nucleoside, or
modifications of
the nucleotide analogues at the 3' or the 5' terminus. For example, the 3'
terminus optionally
include a 3' cationic group, or by inverting the nucleoside at the 3'-terminus
with a 3'-3'
linkage. In another alternative, the 3'-terminus is optionally conjugated with
an aminoalkyl
group, e.g., a 3' C5-aminoalkyl dT. In an additional alternative, the 3'-
terminus is optionally
conjugated with an abasic site, e.g., with an apurinic or apyrimidinic site.
[0082] In some embodiments, the oligonucleotide comprising a compound of
Formula (II)
(e.g., Formula Ha) further comprises one or more of the artificial nucleotide
analogues described
herein. In some instances, the oligonucleotide comprising a compound of
Formula (II) (e.g.,
Formula Ha) further comprises one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15,
16, 17, 18, 20, 25, or more additional modifications such as, but not limited
to, 2'-0-methyl, 2'-
0-methoxyethyl (2'-0-M0E), 2'-0-aminopropyl, 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-
0-
aminopropyl (2'-0-AP), 2'-0-dimethylaminoethyl (2'-0-DMA0E), 2'-0-
dimethylaminopropyl
(2'-0-DMAP), 2'-0- dimethylaminoethyloxyethyl (2'-0-DMAEOE), or 2'-0-N-
methylacetamido (2'-0-NMA) modified, LNA, ENA, PNA, HNA, morpholino,
methylphosphonate nucleotides, thiolphosphonate nucleotides, 2'-fluoro N3-P5'-
phosphoramidites, or a combination thereof In some instances, the
oligonucleotide comprising
a compound of Formula (II) (e.g., Formula Ha) further comprises 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 20, 25, or more of the artificial nucleotide
analogues selected from 2'-
0-methyl, 2'-0-methoxyethyl (2'-0-M0E), 2'-0-aminopropyl, 2'-deoxy, 2-deoxy-2'-
fluoro, 2'-
0-aminopropyl (2'-0-AP), 2'-0-dimethylaminoethyl (2'-0-DMA0E), 2'-0-
dimethylaminopropyl (2'-0-DMAP), 2'-0- dimethylaminoethyloxyethyl (2'-0-
DMAEOE), or 2'-
0-N-methylacetamido (2'-0-NMA) modified, LNA, ENA, PNA, HNA, morpholino,
methylphosphonate nucleotides, thiolphosphonate nucleotides, 2'-fluoro N3-P5'-
phosphoramidites, or a combination thereof In some instances, the
oligonucleotide comprising
a compound of Formula (II) (e.g., Formula Ha) further comprises 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 20, 25, or more of 2'-0-methyl modified
nucleotides. In some
instances, the oligonucleotide comprising a compound of Formula (II) (e.g.,
Formula Ha) further
comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20,
25, or more of 2'-O-
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methoxyethyl (2'-0-M0E) modified nucleotides. In some instances, the
oligonucleotide
comprising a compound of Formula (II) (e.g., Formula ha) further comprises 1,
2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 25, or more of thiolphosphonate
nucleotides.
[0083] In some instances, about 5 to about 100% of the oligonucleotide
comprising a
compound of Formula (II) (e.g., Formula ha) comprise the artificial nucleotide
analogues
described herein. In some instances, about 5%, 10%, 15%, 20%, 25%, 30%, 35%,
40%, 45%,
50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the polynucleic
acid
molecule comprise the artificial nucleotide analogues described herein. In
some embodiments,
the artificial nucleotide analogues include 2' -0-methyl, 2'-0-methoxyethyl
(2'-0-M0E), 2'-0-
aminopropyl, 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-0-aminopropyl (2'-0-AP), 2'-0-
dimethylaminoethyl (2'-0-DMA0E), 2'-0-dimethylaminopropyl (2'-0-DMAP), 2'-0-
dimethylaminoethyloxyethyl (2'-0-DMAEOE), or 2'-0-N-methylacetamido (2'-0-NMA)
modified, LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotides,
thiolphosphonate nucleotides, 2'-fluoro N3-P5'-phosphoramidites, or a
combination thereof
[0084] In some embodiments, the oligonucleotide (or B) described herein
comprises RNA or
DNA. In some cases, the oligonucleotide comprises RNA. In some instances, RNA
comprises
short interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA),
double-
stranded RNA (dsRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), or
heterogeneous
nuclear RNA (hnRNA). In some instances, RNA comprises shRNA. In some
instances, RNA
comprises miRNA. In some instances, RNA comprises dsRNA. In some instances,
RNA
comprises tRNA. In some instances, RNA comprises rRNA. In some instances, RNA
comprises hnRNA. In some instances, the RNA comprises siRNA. In some cases,
the
oligonucleotide comprises a sense strand (or passenger strand) of a siRNA. In
other cases, the
oligonucleotide comprises an antisense (or guide strand) of a siRNA.
[0085] In some embodiments, the oligonucleotide is from about 10 to about 50
nucleotides in
length. In some instances, the oligonucleotide is from about 10 to about 30,
from about 15 to
about 30, from about 18 to about 25, from about 18 to about 24, from about 19
to about 23, or
from about 20 to about 22 nucleotides in length.
[0086] In some embodiments, the oligonucleotide is about 50 nucleotides in
length. In some
instances, the oligonucleotide is about 45 nucleotides in length. In some
instances, the
oligonucleotide is about 40 nucleotides in length. In some instances, the
oligonucleotide is
about 35 nucleotides in length. In some instances, the oligonucleotide is
about 30 nucleotides in
length. In some instances, the oligonucleotide is about 25 nucleotides in
length. In some
instances, the oligonucleotide is about 20 nucleotides in length. In some
instances, the
oligonucleotide is about 19 nucleotides in length. In some instances, the
oligonucleotide is
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about 18 nucleotides in length. In some instances, the oligonucleotide is
about 17 nucleotides in
length. In some instances, the oligonucleotide is about 16 nucleotides in
length. In some
instances, the oligonucleotide is about 15 nucleotides in length. In some
instances, the
oligonucleotide is about 14 nucleotides in length. In some instances, the
oligonucleotide is
about 13 nucleotides in length. In some instances, the oligonucleotide is
about 12 nucleotides in
length. In some instances, the oligonucleotide is about 11 nucleotides in
length. In some
instances, the oligonucleotide is about 10 nucleotides in length. In some
instances, the
oligonucleotide is from about 10 to about 50 nucleotides in length. In some
instances, the
oligonucleotide is from about 10 to about 45 nucleotides in length. In some
instances, the
oligonucleotide is from about 10 to about 40 nucleotides in length. In some
instances, the
oligonucleotide is from about 10 to about 35 nucleotides in length. In some
instances, the
oligonucleotide is from about 10 to about 30 nucleotides in length. In some
instances, the
oligonucleotide is from about 10 to about 25 nucleotides in length. In some
instances, the
oligonucleotide is from about 10 to about 20 nucleotides in length. In some
instances, the
oligonucleotide is from about 15 to about 25 nucleotides in length. In some
instances, the
oligonucleotide is from about 19 to about 23 nucleotides in length. In some
instances, the
oligonucleotide is from about 15 to about 30 nucleotides in length. In some
instances, the
oligonucleotide is from about 12 to about 30 nucleotides in length.
[0087] In some embodiments, the oligonucleotide is further hybridized with a
second
oligonucleotide to form a duplex. In some instances, the oligonucleotide is a
sense strand or
passenger strand. In some instances, the second oligonucleotide is an
antisense strand or guide
strand.
[0088] In some embodiments, the second oligonucleotide is from about 10 to
about 50
nucleotides in length. In some instances, the second oligonucleotide is from
about 10 to about
30, from about 15 to about 30, from about 18 to about 25, from about 18 to
about 24, from about
19 to about 23, or from about 20 to about 22 nucleotides in length.
[0089] In some instances, the second oligonucleotide is about 50 nucleotides
in length. In
some instances, the second oligonucleotide is about 45 nucleotides in length.
In some instances,
the second oligonucleotide is about 40 nucleotides in length. In some
instances, the second
oligonucleotide is about 35 nucleotides in length. In some instances, the
second oligonucleotide
is about 30 nucleotides in length. In some instances, the second
oligonucleotide is about 25
nucleotides in length. In some instances, the second oligonucleotide is about
20 nucleotides in
length. In some instances, the second oligonucleotide is about 19 nucleotides
in length. In some
instances, the second oligonucleotide is about 18 nucleotides in length. In
some instances, the
second oligonucleotide is about 17 nucleotides in length. In some instances,
the second
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oligonucleotide is about 16 nucleotides in length. In some instances, the
second oligonucleotide
is about 15 nucleotides in length. In some instances, the second
oligonucleotide is about 14
nucleotides in length. In some instances, the second oligonucleotide is about
13 nucleotides in
length. In some instances, the second oligonucleotide is about 12 nucleotides
in length. In some
instances, the second oligonucleotide is about 11 nucleotides in length. In
some instances, the
second oligonucleotide is about 10 nucleotides in length. In some instances,
the second
oligonucleotide is from about 10 to about 50 nucleotides in length. In some
instances, the
second oligonucleotide is from about 10 to about 45 nucleotides in length. In
some instances,
the second oligonucleotide is from about 10 to about 40 nucleotides in length.
In some
instances, the second oligonucleotide is from about 10 to about 35 nucleotides
in length. In
some instances, the second oligonucleotide is from about 10 to about 30
nucleotides in length.
In some instances, the second oligonucleotide is from about 10 to about 25
nucleotides in length.
In some instances, the second oligonucleotide is from about 10 to about 20
nucleotides in length.
In some instances, the second oligonucleotide is from about 15 to about 25
nucleotides in length.
In some instances, the second oligonucleotide is from about 15 to about 25
nucleotides in length.
In some instances, the second oligonucleotide is from about 15 to about 30
nucleotides in length.
In some instances, the second oligonucleotide is from about 12 to about 30
nucleotides in length.
[0090] In some embodiments, a polynucleic acid molecule comprises a first
oligonucleotide
and a second oligonucleotide. In some instances, the polynucleic acid molecule
further
comprises a blunt terminus, an overhang, or a combination thereof In some
instances, the blunt
terminus is a 5' blunt terminus, a 3' blunt terminus, or both. In some cases,
the overhang is a 5'
overhang, 3' overhang, or both. In some cases, the overhang comprises 1, 2, 3,
4, 5, 6, 7, 8, 9, or
non-base pairing nucleotides. In some cases, the overhang comprises 1, 2, 3,
4, 5, or 6 non-
base pairing nucleotides. In some cases, the overhang comprises 1, 2, 3, or 4
non-base pairing
nucleotides. In some cases, the overhang comprises 1 non-base pairing
nucleotide. In some
cases, the overhang comprises 2 non-base pairing nucleotides. In some cases,
the overhang
comprises 3 non-base pairing nucleotides. In some cases, the overhang
comprises 4 non-base
pairing nucleotides.
[0091] In some embodiments, the sequence of the polynucleic acid molecule is
at least 40%,
50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 99.5%
complementary
to a target sequence described herein. Exemplary target sequence includes, but
not limited to, any
sequences of DMD gene or its mRNA, DMPK gene or its mRNA, any sequences of an
oncogene or its
mRNA, any genes related to hereditary or genetic diseases (e.g., GYS1), any
sequences of a gene related
to muscle atrophy, muscle dystrophy, or muscle wasting (e.g., DMD, DMPK, DUX4)
and its mRNA. In
some embodiments, the sequence of the polynucleic acid molecule is at least
50%
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complementary to a target sequence described herein. In some embodiments, the
sequence of
the polynucleic acid molecule is at least 60% complementary to a target
sequence described
herein. In some embodiments, the sequence of the polynucleic acid molecule is
at least 70%
complementary to a target sequence described herein. In some embodiments, the
sequence of
the polynucleic acid molecule is at least 80% complementary to a target
sequence described
herein. In some embodiments, the sequence of the polynucleic acid molecule is
at least 90%
complementary to a target sequence described herein. In some embodiments, the
sequence of
the polynucleic acid molecule is at least 95% complementary to a target
sequence described
herein. In some embodiments, the sequence of the polynucleic acid molecule is
at least 99%
complementary to a target sequence described herein. In some instances, the
sequence of the
polynucleic acid molecule is 100% complementary to a target sequence described
herein.
[0092] In some embodiments, the sequence of the polynucleic acid molecule has
5 or less
mismatches to a target sequence described herein. In some embodiments, the
sequence of the
polynucleic acid molecule has 4 or less mismatches to a target sequence
described herein. In
some instances, the sequence of the polynucleic acid molecule has 3 or less
mismatches to a
target sequence described herein. In some cases, the sequence of the
polynucleic acid molecule
has 2 or less mismatches to a target sequence described herein. In some cases,
the sequence of
the polynucleic acid molecule has 1 or less mismatches to a target sequence
described herein.
[0093] In some embodiments, the specificity of the polynucleic acid molecule
that hybridizes
to a target sequence described herein is a 95%, 98%, 99%, 99.5%, or 100%
sequence
complementarity of the polynucleic acid molecule to a target sequence. In some
instances, the
hybridization is a high stringent hybridization condition.
[0094] In some embodiments, the polynucleic acid molecule hybridizes to at
least 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, 20, or more contiguous bases of a target
sequence described
herein. In some embodiments, the polynucleic acid molecule hybridizes to at
least 8 contiguous
bases of a target sequence described herein. In some embodiments, the
polynucleic acid
molecule hybridizes to at least 9 contiguous bases of a target sequence
described herein. In some
embodiments, the polynucleic acid molecule hybridizes to at least 10
contiguous bases of a
target sequence described herein. In some embodiments, the polynucleic acid
molecule
hybridizes to at least 11 contiguous bases of a target sequence described
herein. In some
embodiments, the polynucleic acid molecule hybridizes to at least 12
contiguous bases of a
target sequence described herein. In some embodiments, the polynucleic acid
molecule
hybridizes to at least 13 contiguous bases of a target sequence described
herein. In some
embodiments, the polynucleic acid molecule hybridizes to at least 14
contiguous bases of a
target sequence described herein. In some embodiments, the polynucleic acid
molecule
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hybridizes to at least 15 contiguous bases of a target sequence described
herein. In some
embodiments, the polynucleic acid molecule hybridizes to at least 16
contiguous bases of a
target sequence described herein. In some embodiments, the polynucleic acid
molecule
hybridizes to at least 17 contiguous bases of a target sequence described
herein. In some
embodiments, the polynucleic acid molecule hybridizes to at least 18
contiguous bases of a
target sequence described herein. In some embodiments, the polynucleic acid
molecule
hybridizes to at least 19 contiguous bases of a target sequence described
herein. In some
embodiments, the polynucleic acid molecule hybridizes to at least 20
contiguous bases of a
target sequence described herein.
[0095] In some embodiments, the polynucleic acid molecule has reduced off-
target effect. In
some instances, "off-target" or "off-target effects" refer to any instance in
which a polynucleic
acid polymer directed against a given target causes an unintended effect by
interacting either
directly or indirectly with another mRNA sequence, a DNA sequence or a
cellular protein or
other moiety. In some instances, an "off-target effect" occurs when there is a
simultaneous
degradation of other transcripts due to partial homology or complementarity
between that other
transcript and the sense and/or antisense strand of the polynucleic acid
molecule.
[0096] In some cases, one or more of the artificial nucleotide analogues
described herein are
resistant toward nucleases such as for example ribonuclease such as RNase H,
deoxyribunuclease such as DNase, or exonuclease such as 5'-3' exonuclease and
3'-5'
exonuclease when compared to natural polynucleic acid molecules. In some
instances, artificial
nucleotide analogues comprising a compound of Formula II (e.g., Formula Ha),
2'-0-methyl, 2'-
0-methoxyethyl (2'-0-M0E), 2'-0-aminopropyl, 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-
0-
aminopropyl (2'-0-AP), 2'-0-dimethylaminoethyl (2'-0-DMA0E), 2'-0-
dimethylaminopropyl
(2'-0-DMAP), 2'-0- dimethylaminoethyloxyethyl (2'-0-DMAEOE), or 2'-0-N-
methylacetamido (2'-0-NMA) modified, LNA, ENA, PNA, HNA, morpholino,
methylphosphonate nucleotides, thiolphosphonate nucleotides, 2'-fluoro N3-P5'-
phosphoramidites, or combinations thereof are resistant toward nucleases such
as for example
ribonuclease such as RNase H, deoxyribunuclease such as DNase, or exonuclease
such as 5' -3'
exonuclease and 3'-5' exonuclease. In some instances, 2'-0-methyl modified
polynucleic acid
molecule is nuclease resistant (e.g., RNase H, DNase, 5'-3' exonuclease or 3'-
5' exonuclease
resistant). In some instances, 2'0-methoxyethyl (2'-0-M0E) modified
polynucleic acid
molecule is nuclease resistant (e.g., RNase H, DNase, 5'-3' exonuclease or 3'-
5' exonuclease
resistant). In some instances, 2'-0-aminopropyl modified polynucleic acid
molecule is nuclease
resistant (e.g., RNase H, DNase, 5'-3' exonuclease or 3'-5' exonuclease
resistant). In some
instances, 2'-deoxy modified polynucleic acid molecule is nuclease resistant
(e.g., RNase H,
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DNase, 5'-3' exonuclease or 3'-5' exonuclease resistant). In some instances,
2'-deoxy-2'-fluoro
modified polynucleic acid molecule is nuclease resistant (e.g., RNase H,
DNase, 5'-3'
exonuclease or 3'-5' exonuclease resistant). In some instances, 2'-0-
aminopropyl (2'-0-AP)
modified polynucleic acid molecule is nuclease resistant (e.g., RNase H,
DNase, 5'-3'
exonuclease or 3'-5' exonuclease resistant). In some instances, 2'-0-
dimethylaminoethyl (2'-0-
DMAOE) modified polynucleic acid molecule is nuclease resistant (e.g., RNase
H, DNase, 5'-3'
exonuclease or 3'-5' exonuclease resistant). In some instances, 2'-0-
dimethylaminopropyl (2'-
0-DMAP) modified polynucleic acid molecule is nuclease resistant (e.g., RNase
H, DNase, 5'-
3' exonuclease or 3'-5' exonuclease resistant). In some instances, 2'-0-
dimethylaminoethyloxyethyl (2'-0-DMAEOE) modified polynucleic acid molecule is
nuclease
resistant (e.g., RNase H, DNase, 5'-3' exonuclease or 3'-5' exonuclease
resistant). In some
instances, 2'-0-N-methylacetamido (2'-0-NMA) modified polynucleic acid
molecule is nuclease
resistant (e.g., RNase H, DNase, 5'-3' exonuclease or 3'-5' exonuclease
resistant). In some
instances, LNA-modified polynucleic acid molecule is nuclease resistant (e.g.,
RNase H, DNase,
5'-3' exonuclease or 3'-5' exonuclease resistant). In some instances, ENA-
modified polynucleic
acid molecule is nuclease resistant (e.g., RNase H, DNase, 5'-3' exonuclease
or 3'-5'
exonuclease resistant). In some instances, HNA-modified polynucleic acid
molecule is nuclease
resistant (e.g., RNase H, DNase, 5'-3' exonuclease or 3'-5' exonuclease
resistant). Morpholinos
may be nuclease resistant (e.g., RNase H, DNase, 5'-3' exonuclease or 3'-5'
exonuclease
resistant). In some instances, PNA-modified polynucleic acid molecule is
resistant to nucleases
(e.g., RNase H, DNase, 5'-3' exonuclease or 3'-5' exonuclease resistant). In
some instances,
methylphosphonate nucleotide-modified polynucleic acid molecule is nuclease
resistant (e.g.,
RNase H, DNase, 5'-3' exonuclease or 3'-5' exonuclease resistant). In some
instances,
thiolphosphonate nucleotide-modified polynucleic acid molecule is nuclease
resistant (e.g.,
RNase H, DNase, 5'-3' exonuclease or 3'-5' exonuclease resistant). In some
instances,
polynucleic acid molecule comprising 2'-fluoro N3-P5'-phosphoramidites is
nuclease resistant
(e.g., RNase H, DNase, 5'-3' exonuclease or 3'-5' exonuclease resistant). in
some instances, the
5' conjugates described herein inhibit 5'-3' exonucleolytic cleavage. In some
instances, the 3'
conjugates described herein inhibit 3'-5' exonucleolytic cleavage.
[0097] In some embodiments, one or more of the artificial nucleotide analogues
comprising a
compound of Formula II (e.g., Formula IIa), described herein have increased
binding affinity
toward their mRNA target relative to an equivalent natural polynucleic acid
molecule. The one
or more of the artificial nucleotide analogues comprising a compound of
Formula II (e.g.,
Formula IIa), 2'-0-methyl, 2'-0-methoxyethyl (2' -0-MOE), 2'-0-aminopropyl, 2'-
deoxy, 2'-
deoxy-2'-fluoro, 2'-0-aminopropyl (2'-0-AP), 2'-0-dimethylaminoethyl (2'-0-
DMA0E), 2'-0-
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dimethylaminopropyl (2'-0-DMAP), 2'-0- dimethylaminoethyloxyethyl (2'-0-
DMAEOE), or 2'-
0-N-methylacetamido (2'-0-NMA) modified, LNA, ENA, PNA, HNA, morpholino,
methylphosphonate nucleotides, thiolphosphonate nucleotides, or 2'-fluoro N3-
P5'-
phosphoramidites can have increased binding affinity toward their mRNA target
relative to an
equivalent natural polynucleic acid molecule. In some instances, 2'-0-methyl
modified
polynucleic acid molecule has increased binding affinity toward their mRNA
target relative to
an equivalent natural polynucleic acid molecule. In some instances, 2'-0-
methoxyethyl (2'-0-
MOE) modified polynucleic acid molecule has increased binding affinity toward
their mRNA
target relative to an equivalent natural polynucleic acid molecule. In some
instances, 2'-0-
aminopropyl modified polynucleic acid molecule has increased binding affinity
toward their
mRNA target relative to an equivalent natural polynucleic acid molecule. In
some instances, 2'-
deoxy modified polynucleic acid molecule has increased binding affinity toward
their mRNA
target relative to an equivalent natural polynucleic acid molecule. In some
instances, 2'-deoxy-
2'-fluoro modified polynucleic acid molecule has increased binding affinity
toward their mRNA
target relative to an equivalent natural polynucleic acid molecule. In some
instances, 2'-0-
aminopropyl (2'-0-AP) modified polynucleic acid molecule has increased binding
affinity
toward their mRNA target relative to an equivalent natural polynucleic acid
molecule. In some
instances, 2'-0-dimethylaminoethyl (2'-0-DMA0E) modified polynucleic acid
molecule has
increased binding affinity toward their mRNA target relative to an equivalent
natural
polynucleic acid molecule. In some instances, 2'-0-dimethylaminopropyl (2'-0-
DMAP)
modified polynucleic acid molecule has increased binding affinity toward their
mRNA target
relative to an equivalent natural polynucleic acid molecule. In some
instances, 2'-0-
dimethylaminoethyloxyethyl (2'-0-DMAEOE) modified polynucleic acid molecule
has
increased binding affinity toward their mRNA target relative to an equivalent
natural
polynucleic acid molecule. In some instances, 2'-0-N-methylacetamido (2'-0-
NMA) modified
polynucleic acid molecule has increased binding affinity toward their mRNA
target relative to
an equivalent natural polynucleic acid molecule. In some instances, LNA-
modified polynucleic
acid molecule has increased binding affinity toward their mRNA target relative
to an equivalent
natural polynucleic acid molecule. In some instances, ENA-modified polynucleic
acid molecule
has increased binding affinity toward their mRNA target relative to an
equivalent natural
polynucleic acid molecule. In some instances, PNA-modified polynucleic acid
molecule has
increased binding affinity toward their mRNA target relative to an equivalent
natural
polynucleic acid molecule. In some instances, HNA-modified polynucleic acid
molecule has
increased binding affinity toward their mRNA target relative to an equivalent
natural
polynucleic acid molecule. In some instances, morpholino-modified polynucleic
acid molecule
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has increased binding affinity toward their mRNA target relative to an
equivalent natural
polynucleic acid molecule. In some instances, methylphosphonate nucleotide-
modified
polynucleic acid molecule has increased binding affinity toward their mRNA
target relative to
an equivalent natural polynucleic acid molecule. In some instances,
thiolphosphonate
nucleotide-modified polynucleic acid molecule has increased binding affinity
toward their
mRNA target relative to an equivalent natural polynucleic acid molecule. In
some instances,
polynucleic acid molecule comprising 2'-fluoro N3-P5'-phosphoramidites has
increased binding
affinity toward their mRNA target relative to an equivalent natural
polynucleic acid molecule. In
some cases, the increased affinity is illustrated with a lower Kd, a higher
melt temperature (Tm),
or a combination thereof
[0098] In some embodiments, an artificial nucleotide analogues comprising a
compound of
Formula II (e.g., Formula Ha), described herein is a chirally pure (or stereo
pure) polynucleic
acid molecule, or a polynucleic acid molecule comprising a single enantiomer.
In some
instances, the polynucleic acid molecule comprises L-nucleotide. In some
instances, the
polynucleic acid molecule comprises D-nucleotides. In some instance, a
polynucleic acid
molecule composition comprises less than 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%,
2%, 1%, or
less of its mirror enantiomer. In some cases, a polynucleic acid molecule
composition
comprises less than 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, or less of a
racemic
mixture. In some instances, the polynucleic acid molecule is a polynucleic
acid molecule
described in: U.S. Patent Publication Nos: 2014/194610 and 2015/211006; and
PCT Publication
No.: W02015107425.
[0099] In some embodiments, a polynucleic acid molecule described herein is
further
modified to include an aptamer-conjugating moiety. In some instances, the
aptamer conjugating
moiety is a DNA aptamer-conjugating moiety. In some instances, the aptamer-
conjugating
moiety is Alphamer (Centauri Therapeutics), which comprises an aptamer portion
that
recognizes a specific cell-surface target and a portion that presents a
specific epitopes for
attaching to circulating antibodies. In some instance, a polynucleic acid
molecule described
herein is further modified to include an aptamer-conjugating moiety as
described in: U.S. Patent
Nos: 8,604,184, 8,591,910, and 7,850,975.
[0100] In additional embodiments, a polynucleic acid molecule described herein
is modified to
increase its stability. In some embodiment, the polynucleic acid molecule is
RNA (e.g., siRNA),
the polynucleic acid molecule is modified to increase its stability. In some
instances, the
polynucleic acid molecule is modified by one or more of the modifications
described above to
increase its stability. In some cases, the polynucleic acid molecule is
modified at the 2' hydroxyl
position, such as by 2'-0-methyl, 2'-0-methoxyethyl (2'-0-M0E), 2'-0-
aminopropyl, 2'-deoxy,
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2'-deoxy-2'-fluoro, 2'-0-aminopropyl (2'-0-AP), 2-0-dimethylaminoethyl (2'-0-
DMA0E), 2'-
0-dimethylaminopropyl (2'-0-DMAP), 2'-0- dimethylaminoethyloxyethyl (2'-0-
DMAEOE), or
2'-0-N-methylacetamido (2'-0-NMA) modification or by a locked or bridged
ribose
conformation (e.g., LNA or ENA). In some cases, the polynucleic acid molecule
is modified by
2'-0-methyl and/or 2'-0-methoxyethyl ribose. In some cases, the polynucleic
acid molecule
also includes morpholinos, PNAs, HNA, methylphosphonate nucleotides,
thiolphosphonate
nucleotides, and/or 2'-fluoro N3-P5'-phosphoramidites to increase its
stability. In some
instances, the polynucleic acid molecule is a chirally pure (or stereo pure)
polynucleic acid
molecule. In some instances, the chirally pure (or stereo pure) polynucleic
acid molecule is
modified to increase its stability. Suitable modifications to the RNA to
increase stability for
delivery will be apparent to the skilled person.
[0101] In some embodiments, a polynucleic acid molecule described herein has
RNAi activity
that modulates expression of RNA encoded by a gene described supra. In some
instances, a
polynucleic acid molecule described herein is a double-stranded siRNA molecule
that down-
regulates expression of a gene, wherein one of the strands of the double-
stranded siRNA
molecule comprises a nucleotide sequence that is complementary to a nucleotide
sequence of the
gene or RNA encoded by the gene or a portion thereof, and wherein the second
strand of the
double-stranded siRNA molecule comprises a nucleotide sequence substantially
similar to the
nucleotide sequence of the gene or RNA encoded by the gene or a portion
thereof. In some
cases, a polynucleic acid molecule described herein is a double-stranded siRNA
molecule that
down-regulates expression of a gene, wherein each strand of the siRNA molecule
comprises
about 15 to 25, 18 to 24, or 19 to about 23 nucleotides, and wherein each
strand comprises at
least about 14, 17, or 19 nucleotides that are complementary to the
nucleotides of the other
strand. In some cases, a polynucleic acid molecule described herein is a
double-stranded siRNA
molecule that down-regulates expression of a gene, wherein each strand of the
siRNA molecule
comprises about 19 to about 23 nucleotides, and wherein each strand comprises
at least about 19
nucleotides that are complementary to the nucleotides of the other strand.
[0102] In some embodiments, a polynucleic acid molecule described herein is
constructed
using chemical synthesis and/or enzymatic ligation reactions using procedures
known in the art.
For example; a polynucleic acid molecule is chemically synthesized using
naturally occurring
nucleotides or variously modified nucleotides designed to increase the
biological stability of the
molecules or to increase the physical stability of the duplex formed between
the polynucleic acid
molecule and target nucleic acids. Exemplary methods include those described
in: U.S. Patent
Nos. 5,142,047; 5,1852444; 5,889,136; 6,008,400; and 6,111,086; PCT
Publication No.
W02009099942; or European Publication No 1579015 Additional exemplary methods
include
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those described in: Griffey et al., "2'-0-aminopropyl ribonudeotides: a
zwitterionic
modification that enhances the exonuclease resistance and biological activity
of antisense
oligonucleotides," J. Med. Chem. 39(26):5100-5109 (1997)), Obika, et al.
"Synthesis of 2'-0,4'-
C-methyleneuridine and -cytidine. Novel bicyclic nucleosides having a fixed
C3, -endo sugar
puckering". Tetrahedron Letters 38 (50): 8735 (1997); Koizumi, M. "ENA
oligonucleotides as
therapeutics". Current opinion in molecular therapeutics 8 (2): 144-149
(2006); and Abramova
et al., "Novel oligonucleotide analogues based on morpholino nucleoside
subunits-antisense
technologies: new chemical possibilities," Indian Journal of Chemistry
48B:1721-1726 (2009).
Alternatively, the polynucleic acid molecule is produced biologically using an
expression vector
into which a polynucleic acid molecule has been subcloned in an antisense
orientation (i.e.,
RNA transcribed from the inserted polynucleic acid molecule will be of an
antisense orientation
to a target polynucleic acid molecule of interest).
[0103] One embodiment provides an oligonucleotide conjugate of Formula (I) or
Formula (I-
A):
A-B
Formula (I);
wherein,
A is a binding moiety;
B is an oligonucleotide comprising a compound of Formula (II) or (ha).
A-B-C
Formula (I-A);
wherein,
A is a binding moiety; and
B is an oligonucleotide comprising a compound of Formula (II) or (ha)
[0104] C is optionally a polymer.One embodiment provides an oligonucleotide
conjugate of
Formula (I) or Formula (I-A):
A-B
Formula (I);
A-B-C
Formula (I-A);
wherein,
A is a binding moiety;
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C is optionally a polymer; and
B is an oligonucleotide comprising a compound of Formula (II-1), (II-2), or
(II-3):
xR3 xL2
0õOjxL2
NC P 0
R2 N R2
R1 41)
(II-1),
R1 (1)
(II-2), or R1
(II-3);
wherein,
R5
HN H
e\r0
iF N WWI
N
Ny NH iõ.(NyNH NyN
Ring G is 0 0 0 , or
NR4R4
=
xis 0, NH, or C(=0);
Ll and L2 are each independently absent or a linking moiety selected from a
phosphodiester, amide, and ester linkage:
R1 is hydrogen, deuterium, halogen, substituted or unsubstituted Ci-Cio alkyl,
substituted
or unsubstituted C2-Cio alkenyl, substituted or unsubstituted C2-Cio alkynyl,
substituted or unsubstituted Ci-Cio fluoroalkyl, substituted or unsubstituted
Ci-C20
heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or
unsubstituted
heterocycloalkyl, substituted or unsubstituted aryl, substituted or
unsubstituted
heteroaryl, substituted or unsubstituted -0-aryl, -0R6, -C(=0)R6, -C(=0)0R6, -
C(=0)NR7R7, -OCH2C(=0)R6, -OCH2C(=0)0R6, or-OCH2C(=0)NR7R7;
each R2 is independently hydrogen, deuterium, or substituted or unsubstituted
Ci-Cio
alkyl;
or two R2 are taken together with the nitrogen atom to which they are attached
to form a
substituted or unsubstituted C3 -C10 heterocycloalkyl;
R3 is hydrogen, 4,4'-dimethoxytrityl (DMT), -R6, -0R6, or -NR7R7;
each R4 is independently hydrogen, substituted or unsubstituted C1-C6alkyl,
substituted
or unsubstituted aryl, or substituted or unsubstituted benzoyl;
R5 is -C(=0)0R6;
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each R6 and R7 are independently hydrogen, substituted or unsubstituted C1-
C6alkyl,
substituted or unsubstituted C1-C6fluoroalkyl, substituted or unsubstituted C3-
C6cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or
unsubstituted aryl, or substituted or unsubstituted heteroaryl.
[0105] In some embodiments of the oligonucleotide conjugate of Formula (I), B
is a
compound having the structure of Formula (11-1). In some embodiments of the
oligonucleotide
conjugate of Formula (I), B is a compound having the structure of Formula (II-
2). In some
embodiments of the oligonucleotide conjugate of Formula B is a compound
having the
structure of Formula (II-3).
[0106] One embodiment provides an oligonucleotide conjugate of :Formula (Xa):
A-X-B'-Y-C
Formula (Xa);
wherein,
A is a binding moiety;
B' is an oligonucleotide comprising compound of Formula (II) or (Ha);
C is optionally a polymer;
X is a bond or a first linker; and
Y is a bond or a second linker;
wherein the polynucleotide further comprises one or more additional non-
natural
nucleotides; and
wherein A and C are not attached to B at the same terminus.
[0107] Another embodiment provides the oligonucleotide comprising a compound
of Formula
(I) or an oligonucleotide conjugate of Formula (Xa), wherein the
oligonucleotide further
comprises, at least one modified internucleotide linkage, or at least one
inverted abasic moiety.
[0108] Another embodiment provides the oligonucleotide comprising a compound
of Formula
(I) or an oligonucleotide conjugate of Formula (Xa), wherein the compound of
Formula II is
located at the 5'-terminus of the oligonucleotide.
[0109] Another embodiment provides the oligonucleotide comprising a compound
of Formula
(I) or an oligonucleotide conjugate of Formula (Xa), wherein the compound of
Formula :lt is
located at an internucleotide linkage of the oligonucleotide.
[0110] Another embodiment provides the oligonucleotide comprising a compound
of Formula
(I) or an oligonucleotide conjugate of Formula (Xa), wherein the compound of
Formula II is
further modified at the 2'-position.
[0111] Another embodiment provides the oligonucleotide comprising a compound
of Formula
(I) or an oligonucleotide conjugate of Formula (Xa), wherein the one or more
additional non-
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natural nucleotides comprises a 2'-modification selected from 2'-0-methyl, 2'-
0-methoxyethyl
(2'-O-M0E), T-deoxy, 2'-deoxy-2-fluoro, 2'-O-aminopropyl (2'-0-AP), 2'-0-
dimethylaminoethyl (2'-O-DNIA0E), 2'-0-dimethylaminopropyl (21-0-DMAP), 2'-0-
dimethylaminoethyloxyethyl (2'-O-DMAEOE), or T-0--N-methylacetamido (2'-O-NMA)
modified nucleotide.
[0112] Another embodiment provides the oligonucleotide comprising a compound
of Formula
(1) or an oligonucleotide conjugate of Formula (Xa), wherein the compound of
Formula II is
selected from locked nucleic acid (LNA) or ethylene nucleic acid (ENA).
[0113] Another embodiment provides the oligonucleotide comprising a compound
of Formula
(I) or an oligonucleotide conjugate of Formula (Xa), wherein the at least one
inverted abasic
moiety is at least one terminus.
[0114] Another embodiment provides the oligonucleotide comprising a compound
of Formula
(1) or an oligonucleotide conjugate of Formula (Xa), wherein the
oligonucleotide is single
stranded. Another embodiment provides the oligonucleotide of Formula (Xa),
wherein the
oligonucleotide is double stranded.
[0115] Another embodiment provides the oligonucleotide comprising a compound
of Formula
(I) or an oligonucleotide conjugate of Formula (Xa), wherein the
oligonucleotide is from 2 to
about 100 residues in length. Another embodiment provides the oligonucleotide
comprising a
compound of Formula (I) or an oligonucleotide conjugate of Formula (Xa),
wherein the
oligonucleotide is from 2 to about 90 residues in length. Another embodiment
provides the
oligonucleotide of Formula (I) or (Xa), wherein the oligonucleotide is from 2
to about 80
residues in length. Another embodiment provides the oligonucleotide of Formula
(I) or (Xa),
wherein the oligonucleotide is from 2 to about 70 residues in length. Another
embodiment
provides the oligonucleotide of Formula (I) or (Xa), wherein the
oligonucleotide is from 2 to
about 60 residues in length. Another embodiment provides the oligonucleotide
of -Formula (I) or
(Xa), wherein the oligonucleotide is from 2 to about 50 residues in length.
Another embodiment
provides the oligonucleotide of Formula (I) or (Xa), wherein the
oligonucleotide is from 2 to
about 40 residues in length. Another embodiment provides the oligonucleotide
of Formula (I) or
(Xa), wherein the oligonucleotide is from 2 to about 30 residues in length.
Another embodiment
provides the oligonucleotide of Formula (I) or (Xa), wherein the
oligonucleotide is from 2 to
about 20 residues in length. Another embodiment provides the oligonucleotide
of Formula (I) or
(Xa), wherein the oligonucleotide is from 2 to about 10 residues in length.
Another embodiment
provides the oligonucleotide of Formula (I) or (Xa), wherein the
oligonucleotide is from 8 to
about 30 residues in length. Another embodiment provides the oligonucleotide
of Formula (Xa),
wherein the oligonucleotide is from 10 to about 30 residues in length. Another
embodiment
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provides the oligonucleotide of Formula (I) or (Xa), wherein the
oligonucleotide is from 14 to
about 30 residues in length. Another embodiment provides the oligonucleotide
of Formula (Xa),
wherein the oligonucleotide is from 18 to about 30 residues in length. Another
embodiment
provides the oligonucleotide of Formula (I) or (Xa), wherein the
oligonucleotide is from 22 to
about 30 residues in length. Another embodiment provides the oligonucleotide
of Formula (I) or
(X), wherein the oligonucleotide is from 26 to about 30 residues in length.
[0116] One embodiment provides a compound suitable for the synthesis of
oligonucleotides
selected from the group:
ODMT e.ro
ODMT ero L,.(N1.(NH
ODMT 1=,O 0
L,O(NyNH 0
,(NerI.iNH 0 9 1 OH
0 Ci) 1 OH 0 N
1 O NC,,) CI (:( H
,P,
0 N
0 N NCõ) (30
NC) )õ 0 , , I LO
I,
H
ODMT nrN
ODMT e.ro
ODMT e.r0
LkON N 0 L0(NyNH L,O(NI.rNH
L 0 0 0
9 1 OH
0 N 0 N 0 N
NC) õL CJI NC.) FIN NC)
N H
INfiN 4.
ODMT e(:)
ODMT e(:)
1
O 0DMT \,0...,NI.rNH I\,ON NH 1\0N1
0 L TO
9 1 F F 0
I 0 e LO
1
0-F 0-ID'N H
0 N-
NC) NC) I NC) )õ Co
----r[qi H
0 1--;_r
ODMT N e-r0
NDMT
Lx0yNyNH
CO
? 1 CO
0 9 N H 0-1:CNI H
NC) 0 ,,)
, or NC o .
Conjugation Chemistry
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[0117] In some embodiments, a polynucleic acid molecule is conjugated to a
binding moiety.
In some instances, the binding moiety comprises amino acids, peptides,
polypeptides, proteins,
antibodies, antigens, toxins, hormones, lipids, nucleotides, nucleosides,
sugars, carbohydrates,
polymers such as polyethylene glycol and polypropylene glycol, as well as
analogs or
derivatives of all of these classes of substances. Additional examples of
binding moiety also
include steroids, such as cholesterol, phospholipids, di-and triacylglycerols,
fatty acids,
hydrocarbons (e.g., saturated, unsaturated, or contains substitutions), enzyme
substrates, biotin,
digoxigenin, and polysaccharides. In some instances, the binding moiety is an
antibody or
binding fragment thereof In some instances, the polynucleic acid molecule is
further
conjugated to a polymer, and optionally an endosomolytic moiety.
[0118] In some embodiments, the polynucleic acid molecule is conjugated to the
binding
moiety by a chemical ligation process. In some instances, the polynucleic acid
molecule is
conjugated to the binding moiety by a native ligation. In some instances, the
conjugation is as
described in: Dawson, et al. "Synthesis of proteins by native chemical
ligation," Science 1994,
266, 776-779; Dawson, et al. "Modulation of Reactivity in Native Chemical
Ligation through
the Use of Thiol Additives," J. Am. Chem. Soc. 1997, 119, 4325-4329; Hackeng,
et al. "Protein
synthesis by native chemical ligation: Expanded scope by using straightforward
methodology.,"
Proc. Natl. Acad. Sci. USA 1999, 96, 10068-10073; or Wu, et al. "Building
complex
glycopeptides: Development of a cysteine-free native chemical ligation
protocol," Angew.
Chem. Int. Ed. 2006, 45, 4116-4125. In some instances, the conjugation is as
described in U.S.
Patent No. 8,936,910. In some embodiments, the polynucleic acid molecule is
conjugated to the
binding moiety either site-specifically or non-specifically via native
ligation chemistry.
[0119] In some instances, the polynucleic acid molecule is conjugated to the
binding moiety
by a site-directed method utilizing a "traceless" coupling technology
(Philochem). In some
instances, the "traceless" coupling technology utilizes an N-terminal 1,2-
aminothiol group on
the binding moiety which is then conjugate with a polynucleic acid molecule
containing an
aldehyde group. (see Casi et at., "Site-specific traceless coupling of potent
cytotoxic drugs to
recombinant antibodies for pharmacodelivery," JACS 134(13): 5887-5892 (2012))
[0120] In some instances, the polynucleic acid molecule is conjugated to the
binding moiety
by a site-directed method utilizing an unnatural amino acid incorporated into
the binding moiety.
In some instances, the unnatural amino acid comprises p-acetylphenylalanine
(pAcPhe). In
some instances, the keto group of pAcPhe is selectively coupled to an alkoxy-
amine derivative
conjugating moiety to form an oxime bond. (see Axup et at., "Synthesis of site-
specific
antibody-drug conjugates using unnatural amino acids," PNAS 109(40): 16101-
16106 (2012)).
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[0121] In some instances, the polynucleic acid molecule is conjugated to the
binding moiety
by a site-directed method utilizing an enzyme-catalyzed process. In some
instances, the site-
directed method utilizes SMARTagTm technology (Redwood). In some instances,
the
SMARTagTm technology comprises generation of a formylglycine (FGly) residue
from cysteine
by formylglycine-generating enzyme (FGE) through an oxidation process under
the presence of
an aldehyde tag and the subsequent conjugation of FGly to an alkylhydraine-
functionalized
polynucleic acid molecule via hydrazino-Pictet-Spengler (HIPS) ligation. (see
Wu et at., "Site-
specific chemical modification of recombinant proteins produced in mammalian
cells by using
the genetically encoded aldehyde tag," PNAS 106(9): 3000-3005 (2009); Agarwal,
et at., "A
Pictet-Spengler ligation for protein chemical modification," PNAS 110(1): 46-
51 (2013)).
[0122] In some instances, the enzyme-catalyzed process comprises microbial
transglutaminase
(mTG). In some cases, the polynucleic acid molecule is conjugated to the
binding moiety
utilizing a microbial transglutaminze catalyzed process. In some instances,
mTG catalyzes the
formation of a covalent bond between the amide side chain of a glutamine
within the recognition
sequence and a primary amine of a functionalized polynucleic acid molecule. In
some instances,
mTG is produced from Streptomyces mobarensis. (see Strop et at., "Location
matters: site of
conjugation modulates stability and pharmacokinetics of antibody drug
conjugates," Chemistry
and Biology 20(2) 161-167 (2013))
[0123] In some instances, the polynucleic acid molecule is conjugated to the
binding moiety
by a method as described in PCT Publication No. W02014/140317, which utilizes
a sequence-
specific transpeptidase.
[0124] In some instances, the polynucleic acid molecule is conjugated to the
binding moiety
by a method as described in U.S. Patent Publication Nos. 2015/0105539 and
2015/0105540.
Binding Moiety
[0125] In some embodiments, the binding moiety A is a polypeptide. In some
instances, the
polypeptide is an antibody or its fragment thereof In some cases, the fragment
is a binding
fragment. In some instances, the antibody or binding fragment thereof
comprises a humanized
antibody or binding fragment thereof, murine antibody or binding fragment
thereof, human
antibody or binding fragment thereof, anti-murine antibody (e.g., anti-mouse
antibody, anti-rat
antibody, etc.), anti-human antibody, chimeric antibody or binding fragment
thereof,
monoclonal antibody or binding fragment thereof, monovalent Fab', divalent
Fab2, F(ab)'3
fragments, single-chain variable fragment (scFv), bis-scFv, (scFv)2, diabody,
minibody,
nanobody, triabody, tetrabody, disulfide stabilized Fv protein (dsFv), single-
domain antibody
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(sdAb), Ig NAR, camelid antibody or binding fragment thereof, bispecific
antibody or biding
fragment thereof, or a chemically modified derivative thereof.
[0126] In some instances, A is an antibody or binding fragment thereof In some
instances, A
is a humanized antibody or binding fragment thereof, murine antibody or
binding fragment
thereof, chimeric antibody or binding fragment thereof, monoclonal antibody or
binding
fragment thereof, monovalent Fab', divalent Fab2, F(ab)'3 fragments, single-
chain variable
fragment (scFv), bis-scFv, (scFv)2, diabody, minibody, nanobody, triabody,
tetrabody, disulfide
stabilized Fv protein ("dsFv"), single-domain antibody (sdAb), Ig NAR, camelid
antibody or
binding fragment thereof, bispecific antibody or biding fragment thereof, or a
chemically
modified derivative thereof In some instances, A is a humanized antibody or
binding fragment
thereof In some instances, A is a murine antibody or binding fragment thereof
In some
instances, A is a chimeric antibody or binding fragment thereof In some
instances, A is a
monoclonal antibody or binding fragment thereof In some instances, A is a full
size antibody.
In some instances, A is a monovalent Fab'. In some instances, A is a divalent
Fab2. In some
instances, A is a single-chain variable fragment (scFv).
[0127] In some embodiments, the binding moiety A is a bispecific antibody or
binding
fragment thereof In some instances, the bispecific antibody is a trifunctional
antibody or a
bispecific mini-antibody. In some cases, the bispecific antibody is a
trifunctional antibody. In
some instances, the trifunctional antibody is a full length monoclonal
antibody comprising
binding sites for two different antigens. Exemplary trifunctional antibodies
include
catumaxomab (which targets EpCAM and CD3; Fresenius Biotech/Trion Pharma),
ertumaxomab (targets HER2/neu/CD3; Fresenius Biotech/Trion Pharma), lymphomun
FBTA05
(targets CD20/CD3; Fresenius Biotech/Trion Pharma), RG7221 (R05520985; targets
Angiopoietin 2/VEGF; Roche), RG7597 (targets Herl/Her3; Genentech/Roche),
M11V1141
(targets IGF1R/Her3; Merrimack), ABT122 (targets TNFa/IL17; Abbvie), ABT981
(targets
IL1a/IL1f3; Abbott), LY3164530 (targets Herl/cMET; Eli Lilly), and TRBS07
(Ektomab; targets
GD2/CD3; Trion Research Gmbh). Additional exemplary trifunctional antibodies
include mAb2
from F-star Biotechnology Ltd. In some instances, A is a bispecific
trifunctional antibody. In
some embodiments, A is a bispecific trifunctional antibody selected from:
catumaxomab (which
targets EpCAM and CD3; Fresenius Biotech/Trion Pharma), ertumaxomab (targets
HER2/neu/CD3; Fresenius Biotech/Trion Pharma), lymphomun FBTA05 (targets
CD20/CD3;
Fresenius Biotech/Trion Pharma), RG7221 (R05520985; targets Angiopoietin
2/VEGF; Roche),
RG7597 (targets Herl/Her3; Genentech/Roche), MM141 (targets IGF1R/Her3;
Merrimack),
ABT122 (targets TNFa/IL17; Abbvie), ABT981 (targets IL1a/IL1f3; Abbott),
LY3164530
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(targets Herl/cMET; Eli Lilly), TRBS07 (Ektomab; targets GD2/CD3; Trion
Research Gmbh),
and a mAb2 from F-star Biotechnology Ltd.
[0128] In some cases, the bispecific antibody is a bispecific mini-antibody.
In some instances,
the bispecific mini-antibody comprises divalent Fab2, F(ab)'3 fragments, bis-
scFv, (scFv)2,
diabody, minibody, triabody, tetrabody or a bi-specific T-cell engager (BiTE).
In some
embodiments, the bi-specific T-cell engager is a fusion protein that contains
two single-chain
variable fragments (scFvs) in which the two scFvs target epitopes of two
different antigens.
Exemplary bispecific mini-antibodies include, but are not limited to, DART
(dual-affinity re-
targeting platform; MacroGenics), blinatumomab (MT103 or AMG103; which targets
CD19/CD3; Micromet), MT111 (targets CEA/CD3; Micromet/Amegen), MT112
(BAY2010112; targets PSMA/CD3; Micromet/Bayer), MT110 (AMG 110; targets
EPCAM/CD3; Amgen/Micromet), MGD006 (targets CD123/CD3; MacroGenics), MGD007
(targets GPA33/CD3; MacroGenics), BI1034020 (targets two different epitopes on
P-amyloid;
Ablynx), ALX0761 (targets IL17A/IL17F; Ablynx), TF2 (targets CEA/hepten;
Immunomedics),
IL-17/IL-34 biAb (BMS), AFM13 (targets CD30/CD16; Affimed), AFM11 (targets
CD19/CD3;
Affimed), and domain antibodies (dAbs from Domantis/GSK).
[0129] In some embodiments, the binding moiety A is a bispecific mini-
antibody. In some
instances, A is a bispecific Fab2. In some instances, A is a bispecific
F(ab)'3 fragment. In some
cases, A is a bispecific bis-scFv. In some cases, A is a bispecific (scFv)2.
In some
embodiments, A is a bispecific diabody. In some embodiments, A is a bispecific
minibody. In
some embodiments, A is a bispecific triabody. In other embodiments, A is a
bispecific
tetrabody. In other embodiments, A is a bi-specific T-cell engager (BiTE). In
additional
embodiments, A is a bispecific mini-antibody selected from: DART (dual-
affinity re-targeting
platform; MacroGenics), blinatumomab (MT103 or AMG103; which targets CD19/CD3;
Micromet), MT111 (targets CEA/CD3; Micromet/Amegen), MT112 (BAY2010112;
targets
PSMA/CD3; Micromet/Bayer), MT110 (AMG 110; targets EPCAM/CD3; Amgen/Micromet),
MGD006 (targets CD123/CD3; MacroGenics), MGD007 (targets GPA33/CD3;
MacroGenics),
BI1034020 (targets two different epitopes on P-amyloid; Ablynx), ALX0761
(targets
IL17A/IL17F; Ablynx), TF2 (targets CEA/hepten; Immunomedics), IL-17/IL-34 biAb
(BMS),
AFM13 (targets CD30/CD16; Affimed), AFM11 (targets CD19/CD3; Affimed), and
domain
antibodies (dAbs from Domantis/GSK).
[0130] In some embodiments, the binding moiety A is a trispecific antibody. In
some
instances, the trispecific antibody comprises F(ab)'3 fragments or a triabody.
In some instances,
A is a trispecific F(ab)'3 fragment. In some cases, A is a triabody. In some
embodiments, A is a
trispecific antibody as described in Dimas, et at., "Development of a
trispecific antibody
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designed to simultaneously and efficiently target three different antigens on
tumor cells," Mol.
Pharmaceutics, 12(9): 3490-3501(2015).
[0131] In some embodiments, the binding moiety A is an antibody or binding
fragment thereof
that recognizes a cell surface protein. In some instances, the cell surface
protein is an antigen
expressed by a cancerous cell. Exemplary cancer antigens include, but are not
limited to, alpha
fetoprotein, ASLG659, B7-H3, BAFF-R, Brevican, CA125 (MUC16), CA15-3, CA19-9,
carcinoembryonic antigen (CEA), CA242, CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1,
teratocarcinoma-derived growth factor), CTLA-4, CXCR5, E16 (LAT1, SLC7A5),
FcRH2
(IFGP4, IRTA4, SPAP1A (SH2 domain containing phosphatase anchor protein la),
SPAP1B,
SPAP1C), epidermal growth factor, ETBR, Fc receptor-like protein 1 (FCRH1),
GEDA, HLA-
DOB (Beta subunit of MHC class II molecule (Ia antigen), human chorionic
gonadotropin,
ICOS, IL-2 receptor, IL20Ra, Immunoglobulin superfamily receptor translocation
associated 2
(IRTA2), L6, Lewis Y, Lewis X, MAGE-1, MAGE-2, MAGE-3, MAGE 4, MARTI,
mesothelin, MDP, MPF (SMR, MSLN), MCP1 (CCL2), macrophage inhibitory factor
(MIF),
MPG, MSG783, mucin, MUC1-KLH, Napi3b (SLC34A2), nectin-4, Neu oncogene
product,
NCA, placental alkaline phosphatase, prostate specific membrane antigen
(PMSA), prostatic
acid phosphatase, PSCA hlg, p9'7, Purinergic receptor P2X ligand-gated ion
channel 5 (P2X5),
LY64 (Lymphocyte antigen 64 (RP105), gp100, P21, six transmembrane epithelial
antigen of
prostate (STEAP1), STEAP2, Sema 5b, tumor-associated glycoprotein 72 (TAG-72),
transferrin
receptor, TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptor
potential cation
channel, subfamily M, member 4) and the like. In some instances, the binding
moiety is an a-
transferrin receptor antibody or binding fragments thereof In some instances,
the binding
moiety is an a-human transferrin receptor antibody. In some instances, the
binding moiety is an
a-human transferrin receptor antibody as described in PCT/US2019/068078, which
is
incorporated by reference herein.
[0132] In some instances, the cell surface protein comprises clusters of
differentiation (CD)
cell surface markers. Exemplary CD cell surface markers include, but are not
limited to, CD1,
CD2, CD3, CD4, CD5, CD6, CD7, CD8, CD9, CD10, CD11a, CD11b, CD11c, CD11d,
CDw12,
CD13, CD14, CD15, CD15s, CD16, CDw17, CD18, CD19, CD20, CD21, CD22, CD23,
CD24,
CD25, CD26, CD27, CD28, CD29, CD30, CD31, CD32, CD33, CD34, CD35, CD36, CD37,
CD38, CD39, CD40, CD41, CD42, CD43, CD44, CD45, CD45RO, CD45RA, CD45RB, CD46,
CD47, CD48, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD50, CD51, CD52, CD53,
CD54, CD55, CD56, CD57, CD58, CD59, CDw60, CD61, CD62E, CD62L (L-selectin),
CD62P, CD63, CD64, CD65, CD66a, CD66b, CD66c, CD66d, CD66e, CD71, CD79 (e.g.,
CD79a, CD79b), CD90, CD95 (Fas), CD103, CD104, CD125 (IL5RA), CD134 (0X40),
CD137
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(4-1BB), CD152 (CTLA-4), CD221, CD274, CD279 (PD-1), CD319 (SLAMF7), CD326
(EpCAM), and the like.
[0133] In some instances, the binding moiety A is an antibody or binding
fragment thereof
that recognizes a cancer antigen. In some instances, the binding moiety A is
an antibody or
binding fragment thereof that recognizes alpha fetoprotein, ASLG659, B7-H3,
BAFF-R,
Brevican, CA125 (MUC16), CA15-3, CA19-9, carcinoembryonic antigen (CEA),
CA242,
CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, teratocarcinoma-derived growth factor),
CTLA-
4, CXCR5, El6 (LAT1, SLC7A5), FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain
containing
phosphatase anchor protein la), SPAP1B, SPAP1C), epidermal growth factor,
ETBR, Fc
receptor-like protein 1 (FCRH1), GEDA, HLA-DOB (Beta subunit of MHC class II
molecule
(Ia antigen), human chorionic gonadotropin, ICOS, IL-2 receptor, IL2ORa,
Immunoglobulin
superfamily receptor translocation associated 2 (IRTA2), L6, Lewis Y, Lewis X,
MAGE-1,
MAGE-2, MAGE-3, MAGE 4, MARTI, mesothelin, MCP1 (CCL2), MDP, macrophage
inhibitory factor (MIF), MPF (SMR, MSLN), MPG, MSG783, mucin, MUC1-KLH, Napi3b
(SLC34A2), nectin-4, Neu oncogene product, NCA, placental alkaline
phosphatase, prostate
specific membrane antigen (PMSA), prostatic acid phosphatase, PSCA hlg, p9'7,
Purinergic
receptor P2X ligand-gated ion channel 5 (P2X5), LY64 (Lymphocyte antigen 64
(RP105),
gp100, P21, six transmembrane epithelial antigen of prostate (STEAP1), STEAP2,
Sema 5b,
tumor-associated glycoprotein 72 (TAG-72), TrpM4 (BR22450, F1120041, TRPM4,
TRPM4B,
transient receptor potential cation channel, subfamily M, member 4) or a
combination thereof
[0134] In some instances, the binding moiety A is an antibody or binding
fragment thereof
that recognizes a CD cell surface marker. In some instances, the binding
moiety A is an
antibody or binding fragment thereof that recognizes CD1, CD2, CD3, CD4, CD5,
CD6, CD7,
CD8, CD9, CD10, CD11a, CD11b, CD11c, CD11d, CDw12, CD13, CD14, CD15, CD15s,
CD16, CDw17, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD28,
CD29, CD30, CD31, CD32, CD33, CD34, CD35, CD36, CD37, CD38, CD39, CD40, CD41,
CD42, CD43, CD44, CD45, CD45RO, CD45RA, CD45RB, CD46, CD47, CD48, CD49a,
CD49b, CD49c, CD49d, CD49e, CD49f, CD50, CD51, CD52, CD53, CD54, CD55, CD56,
CD57, CD58, CD59, CDw60, CD61, CD62E, CD62L (L-selectin), CD62P, CD63, CD64,
CD65, CD66a, CD66b, CD66c, CD66d, CD66e, CD71, CD79 (e.g., CD79a, CD79b),
CD90,
CD95 (Fas), CD103, CD104, CD125 (IL5RA), CD134 (0X40), CD137 (4-1BB), CD152
(CTLA-4), CD221, CD274, CD279 (PD-1), CD319 (SLAMF7), CD326 (EpCAM), or a
combination thereof
[0135] In some embodiments, the antibody or binding fragment thereof comprises
zalutumumab (HuMax-EFGr, Genmab), abagovomab (Menarini), abituzumab (Merck),
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adecatumumab (MT201), alacizumab pegol, alemtuzumab (Campath , MabCampath, or
Campath-1H; Leukosite), AlloMune (BioTransplant), amatuximab (Morphotek,
Inc.), anti-
VEGF (Genetech), anatumomab mafenatox, apolizumab (hulD10), ascrinvacumab
(Pfizer Inc.),
atezolizumab (MPDL3280A; Genentech/Roche), B43.13 (OvaRex, AltaRex
Corporation),
basiliximab (Simulect , Novartis), belimumab (Benlysta , GlaxoSmithKline),
bevacizumab
(Avasting, Genentech), blinatumomab (Blincyto, AMG103; Amgen), BEC2 (ImGlone
Systems
Inc.), carlumab (Janssen Biotech), catumaxomab (Removab, Trion Pharma),
CEAcide
(Immunomedics), Cetuximab (Erbitux , ImClone), citatuzumab bogatox (VB6-845),
cixutumumab (IMC-Al2, ImClone Systems Inc.), conatumumab (AMG 655, Amgen),
dacetuzumab (SGN-40, huS2C6; Seattle Genetics, Inc.), daratumumab (Darzalex ,
Janssen
Biotech), detumomab, drozitumab (Genentech), durvalumab (MedImmune),
dusigitumab
(MedImmune), edrecolomab (MAb17-1A, Panorex, Glaxo Wellcome), elotuzumab
(EmplicitiTM, Bristol-Myers Squibb), emibetuzumab (Eli Lilly), enavatuzumab
(Facet Biotech
Corp.), enfortumab vedotin (Seattle Genetics, Inc.), enoblituzumab (MGA271,
MacroGenics,
Inc.), ensituxumab (Neogenix Oncology, Inc.), epratuzumab (LymphoCide,
Immunomedics,
Inc.), ertumaxomab (Rexomung, Trion Pharma), etaracizumab (Abegrin,
MedImmune),
farletuzumab (MORAb-003, Morphotek, Inc), FBTA05 (Lymphomun, Trion Pharma),
ficlatuzumab (AVEC, Pharmaceuticals), figitumumab (CP-751871, Pfizer),
flanvotumab
(ImClone Systems), fresolimumab (GC1008, Aanofi-Aventis), futuximab, glaximab,
ganitumab
(Amgen), girentuximab (Rencarex , Wilex AG), IMAB362 (Claudiximab, Ganymed
Pharmaceuticals AG), imalumab (Baxalta), IMC-1C11 (ImClone Systems), IMC-C225
(Imclone
Systems Inc.), imgatuzumab (Genentech/Roche), intetumumab (Centocor, Inc.),
ipilimumab
(Yervoy , Bristol-Myers Squibb), iratumumab (Medarex, Inc.), isatuximab
(5AR650984,
Sanofi-Aventis), labetuzumab (CEA-CIDE, Immunomedics), lexatumumab (ETR2-ST01,
Cambridge Antibody Technology), lintuzumab (SGN-33, Seattle Genetics),
lucatumumab
(Novartis), lumiliximab, mapatumumab (HGS-ETR1, Human Genome Sciences),
matuzumab
(EMD 72000, Merck), milatuzumab (hLL1, Immunomedics, Inc.), mitumomab (BEC-2,
ImClone Systems), narnatumab (ImClone Systems), necitumumab (PortrazzaTM, Eli
Lilly),
nesvacumab (Regeneron Pharmaceuticals), nimotuzumab (h-R3, BIOMAb EGFR,
TheraCIM,
Theraloc, or CIMAher; Biotech Pharmaceutical Co.), nivolumab (Opdivo , Bristol-
Myers
Squibb), obinutuzumab (Gazyva or Gazyvaro; Hoffmann-La Roche), ocaratuzumab
(AME-
133v, LY2469298; Mentrik Biotech, LLC), ofatumumab (Arzerra , Genmab),
onartuzumab
(Genentech), Ontuxizumab (Morphotek, Inc.), oregovomab (OvaRex , AltaRex
Corp.),
otlertuzumab (Emergent BioSolutions), panitumumab (ABX-EGF, Amgen), pankomab
(Glycotope GMBH), parsatuzumab (Genentech), patritumab, pembrolizumab
(Keytruda ,
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Merck), pemtumomab (Theragyn, Antisoma), pertuzumab (Perjeta, Genentech),
pidilizumab
(CT-011, Medivation), polatuzumab vedotin (Genentech/Roche), pritumumab,
racotumomab
(Vaxira , Recombio), ramucirumab (Cyramza , ImClone Systems Inc.), rituximab
(Rituxan ,
Genentech), robatumumab (Schering-Plough), Seribantumab (Sanofi/Merrimack
Pharmaceuticals, Inc.), sibrotuzumab, siltuximab (SylvantTM, Janssen Biotech),
Smart MI95
(Protein Design Labs, Inc.), Smart ID10 (Protein Design Labs, Inc.), tabalumab
(LY2127399,
Eli Lilly), taplitumomab paptox, tenatumomab, teprotumumab (Roche), tetulomab,
TGN1412
(CD28-SuperMAB or TAB08), tigatuzumab (CD-1008, Daiichi Sankyo), tositumomab,
trastuzumab (Hercepting), tremelimumab (CP-672,206; Pfizer), tucotuzumab
celmoleukin
(EMD Pharmaceuticals), ublituximab, urelumab (BMS-663513, Bristol-Myers
Squibb),
volociximab (M200, Biogen Idec), zatuximab, and the like.
[0136] In some embodiments, the binding moiety A comprises zalutumumab (HuMax-
EFGr,
Genmab), abagovomab (Menarini), abituzumab (Merck), adecatumumab (MT201),
alacizumab
pegol, alemtuzumab (Campath , MabCampath, or Campath-1H; Leukosite), AlloMune
(BioTransplant), amatuximab (Morphotek, Inc.), anti-VEGF (Genetech),
anatumomab
mafenatox, apolizumab (hulD10), ascrinvacumab (Pfizer Inc.), atezolizumab
(MPDL3280A;
Genentech/Roche), B43.13 (OvaRex, AltaRex Corporation), basiliximab (Simulect
, Novartis),
belimumab (Benlysta , GlaxoSmithKline), bevacizumab (Avasting, Genentech),
blinatumomab (Blincyto, AMG103; Amgen), BEC2 (ImGlone Systems Inc.), carlumab
(Janssen
Biotech), catumaxomab (Removab, Trion Pharma), CEAcide (Immunomedics),
Cetuximab
(Erbitux , ImClone), citatuzumab bogatox (VB6-845), cixutumumab (IMC-Al2,
ImClone
Systems Inc.), conatumumab (AMG 655, Amgen), dacetuzumab (SGN-40, huS2C6;
Seattle
Genetics, Inc.), daratumumab (Darzalex , Janssen Biotech), detumomab,
drozitumab
(Genentech), durvalumab (MedImmune), dusigitumab (MedImmune), edrecolomab
(MAb17-
1A, Panorex, Glaxo Wellcome), elotuzumab (EmplicitiTM, Bristol-Myers Squibb),
emibetuzumab (Eli Lilly), enavatuzumab (Facet Biotech Corp.), enfortumab
vedotin (Seattle
Genetics, Inc.), enoblituzumab (MGA271, MacroGenics, Inc.), ensituxumab
(Neogenix
Oncology, Inc.), epratuzumab (LymphoCide, Immunomedics, Inc.), ertumaxomab
(Rexomung,
Trion Pharma), etaracizumab (Abegrin, MedImmune), farletuzumab (MORAb-003,
Morphotek,
Inc), FBTA05 (Lymphomun, Trion Pharma), ficlatuzumab (AVEC, Pharmaceuticals),
figitumumab (CP-751871, Pfizer), flanvotumab (ImClone Systems), fresolimumab
(GC1008,
Aanofi-Aventis), futuximab, glaximab, ganitumab (Amgen), girentuximab
(Rencarex , Wilex
AG), IMAB362 (Claudiximab, Ganymed Pharmaceuticals AG), imalumab (Baxalta),
IMC-1C11
(ImClone Systems), IMC-C225 (Imclone Systems Inc.), imgatuzumab
(Genentech/Roche),
intetumumab (Centocor, Inc.), ipilimumab (Yervoy , Bristol-Myers Squibb),
iratumumab
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(Medarex, Inc.), isatuximab (SAR650984, Sanofi-Aventis), labetuzumab (CEA-
CIDE,
Immunomedics), lexatumumab (ETR2-ST01, Cambridge Antibody Technology),
lintuzumab
(SGN-33, Seattle Genetics), lucatumumab (Novartis), lumiliximab, mapatumumab
(HGS-ETR1,
Human Genome Sciences), matuzumab (EMD 72000, Merck), milatuzumab (hLL1,
Immunomedics, Inc.), mitumomab (BEC-2, ImClone Systems), narnatumab (ImClone
Systems),
necitumumab (PortrazzaTM, Eli Lilly), nesvacumab (Regeneron Pharmaceuticals),
nimotuzumab
(h-R3, BIOMAb EGFR, TheraCIM, Theraloc, or CIMAher; Biotech Pharmaceutical
Co.),
nivolumab (Opdivog, Bristol-Myers Squibb), obinutuzumab (Gazyva or Gazyvaro;
Hoffmann-
La Roche), ocaratuzumab (AME-133v, LY2469298; Mentrik Biotech, LLC),
ofatumumab
(Arzerrag, Genmab), onartuzumab (Genentech), Ontuxizumab (Morphotek, Inc.),
oregovomab
(OvaRex , AltaRex Corp.), otlertuzumab (Emergent BioSolutions), panitumumab
(ABX-EGF,
Amgen), pankomab (Glycotope GMBH), parsatuzumab (Genentech), patritumab,
pembrolizumab (Keytrudag, Merck), pemtumomab (Theragyn, Antisoma), pertuzumab
(Perj eta,
Genentech), pidilizumab (CT-011, Medivation), polatuzumab vedotin
(Genentech/Roche),
pritumumab, racotumomab (Vaxirag, Recombio), ramucirumab (Cyramzag, ImClone
Systems
Inc.), rituximab (Rituxang, Genentech), robatumumab (Schering-Plough),
Seribantumab
(Sanofi/Merrimack Pharmaceuticals, Inc.), sibrotuzumab, siltuximab (SylvantTM,
Janssen
Biotech), Smart MI95 (Protein Design Labs, Inc.), Smart ID10 (Protein Design
Labs, Inc.),
tabalumab (LY2127399, Eli Lilly), taplitumomab paptox, tenatumomab,
teprotumumab (Roche),
tetulomab, TGN1412 (CD28-SuperMAB or TAB08), tigatuzumab (CD-1008, Daiichi
Sankyo),
tositumomab, trastuzumab (Hercepting), tremelimumab (CP-672,206; Pfizer),
tucotuzumab
celmoleukin (EMD Pharmaceuticals), ublituximab, urelumab (BMS-663513, Bristol-
Myers
Squibb), volociximab (M200, Biogen Idec), or zatuximab. In some embodiments,
the binding
moiety A is zalutumumab (HuMax-EFGr, by Genmab).
Additional Binding Moieties
[0137] In some embodiments, the binding moiety is a plasma protein. In some
instances, the
plasma protein comprises albumin. In some instances, the binding moiety A is
albumin. In
some instances, albumin is conjugated by one or more of a conjugation
chemistry described
herein to a polynucleic acid molecule. In some instances, albumin is
conjugated by native
ligation chemistry to a polynucleic acid molecule. In some instances, albumin
is conjugated by
lysine conjugation to a polynucleic acid molecule.
[0138] In some instances, the binding moiety is a steroid. Exemplary steroids
include
cholesterol, phospholipids, di-and triacylglycerols, fatty acids, hydrocarbons
that are saturated,
unsaturated, comprise substitutions, or combinations thereof In some
instances, the steroid is
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cholesterol. In some instances, the binding moiety is cholesterol. In some
instances, cholesterol
is conjugated by one or more of a conjugation chemistry described herein to a
polynucleic acid
molecule. In some instances, cholesterol is conjugated by native ligation
chemistry to a
polynucleic acid molecule. In some instances, cholesterol is conjugated by
lysine conjugation to
a polynucleic acid molecule.
[0139] In some instances, the binding moiety is a polymer, including but not
limited to poly
nucleic acid molecule aptamers that bind to specific surface markers on cells.
In this instance the
binding moiety is a polynucleic acid that does not hybridize to a target gene
or mRNA, but
instead is capable of selectively binding to a cell surface marker similarly
to an antibody binding
to its specific epitope of a cell surface marker.
[0140] In some cases, the binding moiety is a peptide. In some cases, the
peptide comprises
between about 1 and about 3 kDa. In some cases, the peptide comprises between
about 1.2 and
about 2.8 kDa, about 1.5 and about 2.5 kDa, or about 1.5 and about 2 kDa. In
some instances,
the peptide is a bicyclic peptide. In some cases, the bicyclic peptide is a
constrained bicyclic
peptide. In some instances, the binding moiety is a bicyclic peptide (e.g.,
bicycles from Bicycle
Therapeutics).
In additional cases, the binding moiety is a small molecule. In some
instances, the small
molecule is an antibody-recruiting small molecule. In some cases, the antibody-
recruiting small
molecule comprises a target-binding terminus and an antibody-binding terminus,
in which the
target-binding terminus is capable of recognizing and interacting with a cell
surface receptor.
For example, in some instances, the target-binding terminus comprising a
glutamate urea
compound enables interaction with PSMA, thereby, enhances an antibody
interaction with a cell
(e.g., a cancerous cell) that expresses PSMA. In some instances, a binding
moiety is a small
molecule described in Zhang et al., "A remote arene-binding site on prostate
specific membrane
antigen revealed by antibody-recruiting small molecules," J Am Chem Soc.
132(36): 12711-
12716 (2010); or McEnaney, et al., "Antibody-recruiting molecules: an emerging
paradigm for
engaging immune function in treating human disease," ACS Chem Biol. 7(7): 1139-
1151
(2012).
Polynucleic Acid Molecule Targets
[0141] In some embodiments, the polynucleic acid molecule B is a polynucleic
acid molecule
(or polynucleotide) that hybridizes to a target region on an oncogene. In some
instances,
oncogenes are further classified into several categories: growth factors or
mitogens, receptor
tyrosine kinases, cytoplasmic tyrosine kinases, cytoplasmic serine/threonine
kinases, regulatory
GTPases, and transcription factors. Exemplary growth factors include c-Sis.
Exemplary
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receptor tyrosine kinases include epidermal growth factor receptor (EGFR),
platelet-derived
growth factor receptor (PDGFR), vascular endothelial growth factor receptor
(VEGFR), and
HER2/neu. Exemplary cytoplasmic tyrosine kinases include Src-family tyrosine
kinases, Syk-
ZAP-70 family of tyrosine kinases, BTK family of tyrosine kinases, and Abl
gene in CML.
Exemplary cytoplasmic serine/threonine kinases include Raf kinase and cyclin-
dependent
kinases. Exemplary regulatory GTPases include Ras family of proteins such as
KRAS.
Exemplary transcription factors include MYC gene. In some instances, an
oncogene described
herein comprises an oncogene selected from growth factors or mitogens,
receptor tyrosine
kinases, cytoplasmic tyrosine kinases, cytoplasmic serine/threonine kinases,
regulatory
GTPases, or transcription factors. In some embodiments, the polynucleic acid
molecule is a
polynucleic acid molecule that hybridizes to a target region of an oncogene
selected from
growth factors or mitogens, receptor tyrosine kinases, cytoplasmic tyrosine
kinases, cytoplasmic
serine/threonine kinases, regulatory GTPases, or transcription factors.
[0142] In some embodiments, an oncogene described herein comprises Abl, AKT-2,
ALK,
AML1 (or RUNX1), AR, AXL, BCL-2, 3, 6, BRAF, c-MYC, EGFR, ErbB-2 (Her2, Neu),
Fms,
FOS, GLI1, HPRT1, IL-3, INTS2, JUN, KIT, KS3, K-sam, LBC (AKAP13), LCK,
Li1101, LA102,
LYL1, MAS1, MDM2, MET, MLL (KAIT2A), MOS, MYB, MYH11/CBFB, NOTCH] (TAN]),
NTRK1 (TRK), OST (SLC51B), PAX5, PIM1, PRAD-1, RAF, RAR/PML, HR/IS, KRAS,
NR/IS,
REL/NRG, RET, ROS, SKI, SRC, TIAM1, or TSC2. In some embodiments, the
polynucleic acid
molecule is a polynucleic acid molecule that hybridizes to a target region of
Abl, AKT-2, ALK,
AML] (or RUNX1), AR, AXL, BCL-2, 3, 6, BRAF, c-MYC, EGFR, ErbB-2 (Her2, Neu),
Fms,
FOS, GUI], HPRT1, IL-3, INTS2, JUN, KIT, KS3, K-sam, LBC (AKAP13), LCK,
Li1101, LA102,
LYL1, MAS1, MDM2, MET, MLL (KAIT2A), MOS, MYB, MYH11/CBFB, NOTCH] (TAN]),
NTRK1 (TRK), OST (SLC51B), PAX5, PIM1, PR/ID-], RAF, RAR/PML, HR/IS, KRAS,
NR/IS,
REL/NRG, RET, ROS, SKI, SRC, TIAM1, or TSC2.
[0143] In some embodiments, an oncogene described herein comprises KRAS, EGFR,
AR,
HPRT1, CNNTB1 (0-catenin), or 13-catenin associated genes. In some
embodiments, the
polynucleic acid molecule B is a polynucleic acid molecule that hybridizes to
a target region of
KRAS, EGFR, AR, HPRT1, CNNTB1 (0-catenin), or 13-catenin associated genes. In
some
embodiments, the polynucleic acid molecule B is a polynucleic acid molecule
that hybridizes to
a target region of KRAS. In some embodiments, the polynucleic acid molecule B
is a
polynucleic acid molecule that hybridizes to a target region of EGFR. In some
embodiments,
the polynucleic acid molecule B is a polynucleic acid molecule that hybridizes
to a target region
of AR. In some embodiments, the polynucleic acid molecule B is a polynucleic
acid molecule
that hybridizes to a target region of CNNTB1 (0-catenin). In some embodiments,
the
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polynucleic acid molecule B is a polynucleic acid molecule that hybridizes to
a target region of
CNNTB1 (0-catenin) associated genes. In some instances, the fl-catenin
associated genes
comprise PIK3CA, PIK3CB, and Myc. In some instances, the polynucleic acid
molecule B is a
polynucleic acid molecule that hybridizes to a target region of HPRT1.
[0144] In some embodiments, the binding moiety A is conjugated according to
Formula (I) to
a polynucleic acid molecule (B), and optionally a polymer (C), described
herein. In some
embodiments, the polynucleic acid molecule comprises a sequence having at
least 50%, 55%,
60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence
identity
to SEQ ID NOs:142-255. 256-369, 481-591, 592-702, 3407-14222, 14222-14238, or
14239-
14352. In some instances, the polynucleic acid molecule comprises a sequence
selected from
SEQ ID NOs: 142-255. 256-369, 481-591, 592-702, 3407-14222, 14222-14238, or
14239-
14352. In some instances, the polymer C optionally comprises polyalkylene
oxide (e.g.,
polyethylene glycol).
[0145] In some embodiments, the binding moiety A is conjugated to a
polynucleic acid
molecule (B), and optionally a polymer (C). In some instances, the binding
moiety A is an
antibody or binding fragment thereof
[0146] In some embodiments, the binding moiety A is conjugated to a
polynucleic acid
molecule (B) non-specifically. In some instances, the binding moiety A is
conjugated to a
polynucleic acid molecule (B) via a lysine residue or a cysteine residue, in a
non-site specific
manner. In some instances, the binding moiety A is conjugated to a polynucleic
acid molecule
(B) via a lysine residue in a non-site specific manner. In some cases, the
binding moiety A is
conjugated to a polynucleic acid molecule (B) via a cysteine residue in a non-
site specific
manner. In some instances, the binding moiety A is an antibody or binding
fragment thereof
[0147] In some embodiments, the binding moiety A is conjugated to a
polynucleic acid
molecule (B) in a site-specific manner. In some instances, the binding moiety
A is conjugated to
a polynucleic acid molecule (B) through a lysine residue, a cysteine residue,
at the 5'-terminus,
at the 3'-terminus, an unnatural amino acid, or an enzyme-modified or enzyme-
catalyzed
residue, via a site-specific manner. In some instances, the binding moiety A
is conjugated to a
polynucleic acid molecule (B) through a lysine residue via a site-specific
manner. In some
instances, the binding moiety A is conjugated to a polynucleic acid molecule
(B) through a
cysteine residue via a site-specific manner. In some instances, the binding
moiety A is
conjugated to a polynucleic acid molecule (B) at the 5'-terminus via a site-
specific manner. In
some instances, the binding moiety A is conjugated to a polynucleic acid
molecule (B) at the 3' -
terminus via a site-specific manner. In some instances, the binding moiety A
is conjugated to a
polynucleic acid molecule (B) through an unnatural amino acid via a site-
specific manner. In
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some instances, the binding moiety A is conjugated to a polynucleic acid
molecule (B) through
an enzyme-modified or enzyme-catalyzed residue via a site-specific manner. In
some instances,
the binding moiety A is an antibody or binding fragment thereof
[0148] In some embodiments, one or more regions of a binding moiety A (e.g.,
an antibody or
binding fragment thereof) is conjugated to a polynucleic acid molecule (B). In
some instances,
the one or more regions of a binding moiety A comprise the N-terminus, the C-
terminus, in the
constant region, at the hinge region, or the Fc region of the binding moiety
A. In some instances,
the polynucleic acid molecule (B) is conjugated to the N-terminus of the
binding moiety A (e.g.,
the N-terminus of an antibody or binding fragment thereof). In some instances,
the polynucleic
acid molecule (B) is conjugated to the C-terminus of the binding moiety A
(e.g., the N-terminus
of an antibody or binding fragment thereof). In some instances, the
polynucleic acid molecule
(B) is conjugated to the constant region of the binding moiety A (e.g., the
constant region of an
antibody or binding fragment thereof). In some instances, the polynucleic acid
molecule (B) is
conjugated to the hinge region of the binding moiety A (e.g., the constant
region of an antibody
or binding fragment thereof). In some instances, the polynucleic acid molecule
(B) is
conjugated to the Fc region of the binding moiety A (e.g., the constant region
of an antibody or
binding fragment thereof).
[0149] In some embodiments, one or more polynucleic acid molecule (B) is
conjugated to a
binding moiety A. In some instances, about 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11,
12, 13, 14, 15, 16, or
more polynucleic acid molecules are conjugated to one binding moiety A. In
some instances,
about 1 polynucleic acid molecule is conjugated to one binding moiety A. In
some instances,
about 2 polynucleic acid molecules are conjugated to one binding moiety A. In
some instances,
about 3 polynucleic acid molecules are conjugated to one binding moiety A. In
some instances,
about 4 polynucleic acid molecules are conjugated to one binding moiety A. In
some instances,
about 5 polynucleic acid molecules are conjugated to one binding moiety A. In
some instances,
about 6 polynucleic acid molecules are conjugated to one binding moiety A. In
some instances,
about 7 polynucleic acid molecules are conjugated to one binding moiety A. In
some instances,
about 8 polynucleic acid molecules are conjugated to one binding moiety A. In
some instances,
about 9 polynucleic acid molecules are conjugated to one binding moiety A. In
some instances,
about 10 polynucleic acid molecules are conjugated to one binding moiety A. In
some
instances, about 11 polynucleic acid molecules are conjugated to one binding
moiety A. In
some instances, about 12 polynucleic acid molecules are conjugated to one
binding moiety A.
In some instances, about 13 polynucleic acid molecules are conjugated to one
binding moiety A.
In some instances, about 14 polynucleic acid molecules are conjugated to one
binding moiety A.
In some instances, about 15 polynucleic acid molecules are conjugated to one
binding moiety A.
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In some instances, about 16 polynucleic acid molecules are conjugated to one
binding moiety A.
In some cases, the one or more polynucleic acid molecules are the same. In
other cases, the one
or more polynucleic acid molecules are different. In some instances, the
binding moiety A is an
antibody or binding fragment thereof
[0150] In some embodiments, the number of polynucleic acid molecule (B)
conjugated to a
binding moiety A (e.g., an antibody or binding fragment thereof) forms a
ratio. In some
instances, the ratio is referred to as a DAR (drug-to-antibody) ratio, in
which the drug as referred
to herein is the polynucleic acid molecule (B). In some instances, the DAR
ratio of the
polynucleic acid molecule (B) to binding moiety A is about 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, 12, 13,
14, 15, 16, or greater. In some instances, the DAR ratio of the polynucleic
acid molecule (B) to
binding moiety A is about 1 or greater. In some instances, the DAR ratio of
the polynucleic acid
molecule (B) to binding moiety A is about 2 or greater. In some instances, the
DAR ratio of the
polynucleic acid molecule (B) to binding moiety A is about 3 or greater. In
some instances, the
DAR ratio of the polynucleic acid molecule (B) to binding moiety A is about 4
or greater. In
some instances, the DAR ratio of the polynucleic acid molecule (B) to binding
moiety A is about
or greater. In some instances, the DAR ratio of the polynucleic acid molecule
(B) to binding
moiety A is about 6 or greater. In some instances, the DAR ratio of the
polynucleic acid
molecule (B) to binding moiety A is about 7 or greater. In some instances, the
DAR ratio of the
polynucleic acid molecule (B) to binding moiety A is about 8 or greater. In
some instances, the
DAR ratio of the polynucleic acid molecule (B) to binding moiety A is about 9
or greater. In
some instances, the DAR ratio of the polynucleic acid molecule (B) to binding
moiety A is about
or greater. In some instances, the DAR ratio of the polynucleic acid molecule
(B) to binding
moiety A is about 11 or greater. In some instances, the DAR ratio of the
polynucleic acid
molecule (B) to binding moiety A is about 12 or greater.
[0151] In some instances, the DAR ratio of the polynucleic acid molecule (B)
to binding
moiety A (e.g., an antibody or binding fragment thereof) is about 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, or 16. In some instances, the DAR ratio of the polynucleic
acid molecule (B) to
binding moiety A is about 1. In some instances, the DAR ratio of the
polynucleic acid molecule
(B) to binding moiety A is about 2. In some instances, the DAR ratio of the
polynucleic acid
molecule (B) to binding moiety A is about 3. In some instances, the DAR ratio
of the
polynucleic acid molecule (B) to binding moiety A is about 4. In some
instances, the DAR ratio
of the polynucleic acid molecule (B) to binding moiety A is about 5. In some
instances, the
DAR ratio of the polynucleic acid molecule (B) to binding moiety A is about 6.
In some
instances, the DAR ratio of the polynucleic acid molecule (B) to binding
moiety A is about 7. In
some instances, the DAR ratio of the polynucleic acid molecule (B) to binding
moiety A is about
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8. In some instances, the DAR ratio of the polynucleic acid molecule (B) to
binding moiety A is
about 9. In some instances, the DAR ratio of the polynucleic acid molecule (B)
to binding
moiety A is about 10. In some instances, the DAR ratio of the polynucleic acid
molecule (B) to
binding moiety A is about 11. In some instances, the DAR ratio of the
polynucleic acid
molecule (B) to binding moiety A is about 12. In some instances, the DAR ratio
of the
polynucleic acid molecule (B) to binding moiety A is about 13. In some
instances, the DAR
ratio of the polynucleic acid molecule (B) to binding moiety A is about 14. In
some instances,
the DAR ratio of the polynucleic acid molecule (B) to binding moiety A is
about 15. In some
instances, the DAR ratio of the polynucleic acid molecule (B) to binding
moiety A is about 16.
[0152] In some instances, the DAR ratio of the polynucleic acid molecule (B)
to binding
moiety A is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. In some
instances, the DAR
ratio of the polynucleic acid molecule (B) to binding moiety A is 1. In some
instances, the DAR
ratio of the polynucleic acid molecule (B) to binding moiety A is 2. In some
instances, the DAR
ratio of the polynucleic acid molecule (B) to binding moiety A is 4. In some
instances, the DAR
ratio of the polynucleic acid molecule (B) to binding moiety A is 6. In some
instances, the DAR
ratio of the polynucleic acid molecule (B) to binding moiety A is 8. In some
instances, the DAR
ratio of the polynucleic acid molecule (B) to binding moiety A is 12.
[0153] In some embodiments, an antibody or its binding fragment is further
modified using
conventional techniques known in the art, for example, by using amino acid
deletion, insertion,
substitution, addition, and/or by recombination and/or any other modification
(e.g.
posttranslational and chemical modifications, such as glycosylation and
phosphorylation) known
in the art either alone or in combination. In some instances, the modification
further comprises a
modification for modulating interaction with Fc receptors. In some instances,
the one or more
modifications include those described in, for example, International
Publication No.
W097/34631, which discloses amino acid residues involved in the interaction
between the Fc
domain and the FcRn receptor. Methods for introducing such modifications in
the nucleic acid
sequence underlying the amino acid sequence of an antibody or its binding
fragment is well
known to the person skilled in the art.
[0154] In some instances, an antibody binding fragment further encompasses its
derivatives
and includes polypeptide sequences containing at least one CDR.
[0155] In some instances, the term "single-chain" as used herein means that
the first and
second domains of a bi-specific single chain construct are covalently linked,
preferably in the
form of a co-linear amino acid sequence encodable by a single nucleic acid
molecule.
[0156] In some instances, a bispecific single chain antibody construct relates
to a construct
comprising two antibody derived binding domains. In such embodiments, bi-
specific single
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chain antibody construct is tandem bi-scFv or diabody. In some instances, a
scFv contains a VH
and VL domain connected by a linker peptide. In some instances, linkers are of
a length and
sequence sufficient to ensure that each of the first and second domains can,
independently from
one another, retain their differential binding specificities.
[0157] In some embodiments, binding to or interacting with as used herein
defines a
binding/interaction of at least two antigen-interaction-sites with each other.
In some instances,
antigen-interaction-site defines a motif of a polypeptide that shows the
capacity of specific
interaction with a specific antigen or a specific group of antigens. In some
cases, the
binding/interaction is also understood to define a specific recognition. In
such cases, specific
recognition refers to that the antibody or its binding fragment is capable of
specifically
interacting with and/or binding to at least two amino acids of each of a
target molecule. For
example, specific recognition relates to the specificity of the antibody
molecule, or to its ability
to discriminate between the specific regions of a target molecule. In
additional instances, the
specific interaction of the antigen-interaction-site with its specific antigen
results in an initiation
of a signal, e.g. due to the induction of a change of the conformation of the
antigen, an
oligomerization of the antigen, etc. In further embodiments, the binding is
exemplified by the
specificity of a "key-lock-principle". Thus in some instances, specific motifs
in the amino acid
sequence of the antigen-interaction-site and the antigen bind to each other as
a result of their
primary, secondary or tertiary structure as well as the result of secondary
modifications of said
structure. In such cases, the specific interaction of the antigen-interaction-
site with its specific
antigen results as well in a simple binding of the site to the antigen.
[0158] In some instances, specific interaction further refers to a reduced
cross-reactivity of the
antibody or its binding fragment or a reduced off-target effect. For example,
the antibody or its
binding fragment that bind to the polypeptide/protein of interest but do not
or do not essentially
bind to any of the other polypeptides are considered as specific for the
polypeptide/protein of
interest. Examples for the specific interaction of an antigen-interaction-site
with a specific
antigen comprise the specificity of a ligand for its receptor, for example,
the interaction of an
antigenic determinant (epitope) with the antigenic binding site of an
antibody.
Production of Antibodies or Binding Fragments Thereof
[0159] In some embodiments, polypeptides described herein (e.g., antibodies
and its binding
fragments) are produced using any method known in the art to be useful for the
synthesis of
polypeptides (e.g., antibodies), in particular, by chemical synthesis or by
recombinant
expression, and are preferably produced by recombinant expression techniques.
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[0160] In some instances, an antibody or its binding fragment thereof is
expressed
recombinantly, and the nucleic acid encoding the antibody or its binding
fragment is assembled
from chemically synthesized oligonucleotides (e.g., as described in Kutmeier
et al., 1994,
BioTechniques 17:242), which involves the synthesis of overlapping
oligonucleotides containing
portions of the sequence encoding the antibody, annealing and ligation of
those
oligonucleotides, and then amplification of the ligated oligonucleotides by
PCR.
[0161] Alternatively, a nucleic acid molecule encoding an antibody is
optionally generated
from a suitable source (e.g., an antibody cDNA library, or cDNA library
generated from any
tissue or cells expressing the immunoglobulin) by PCR amplification using
synthetic primers
hybridizable to the 3' and 5' ends of the sequence or by cloning using an
oligonucleotide probe
specific for the particular gene sequence.
[0162] In some instances, an antibody or its binding is optionally generated
by immunizing an
animal, such as a rabbit, to generate polyclonal antibodies or, more
preferably, by generating
monoclonal antibodies, e.g., as described by Kohler and Milstein (1975, Nature
256:495-497)
or, as described by Kozbor et al. (1983, Immunology Today 4:72) or Cole et al.
(1985 in
Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).
Alternatively, a
clone encoding at least the Fab portion of the antibody is optionally obtained
by screening Fab
expression libraries (e.g., as described in Huse et al., 1989, Science
246:1275-1281) for clones
of Fab fragments that bind the specific antigen or by screening antibody
libraries (See, e.g.,
Clackson et al., 1991, Nature 352:624; Hane et al., 1997 Proc. Natl. Acad.
Sci. USA 94:4937).
[0163] In some embodiments, techniques developed for the production of
"chimeric
antibodies" (Morrison et al., 1984, Proc. Natl. Acad. Sci. 81:851-855;
Neuberger et al., 1984,
Nature 312:604-608; Takeda et al., 1985, Nature 314:452-454) by splicing genes
from a mouse
antibody molecule of appropriate antigen specificity together with genes from
a human antibody
molecule of appropriate biological activity are used. A chimeric antibody is a
molecule in which
different portions are derived from different animal species, such as those
having a variable
region derived from a murine monoclonal antibody and a human immunoglobulin
constant
region, e.g., humanized antibodies.
[0164] In some embodiments, techniques described for the production of single
chain
antibodies (U.S. Pat. No. 4,694,778; Bird, 1988, Science 242:423-42; Huston et
al., 1988, Proc.
Natl. Acad. Sci. USA 85:5879-5883; and Ward et al., 1989, Nature 334:544-54)
are adapted to
produce single chain antibodies. Single chain antibodies are formed by linking
the heavy and
light chain fragments of the Fv region via an amino acid bridge, resulting in
a single chain
polypeptide. Techniques for the assembly of functional Fv fragments in E. coli
are also
optionally used (Skerra et al., 1988, Science 242:1038-1041).
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[0165] In some embodiments, an expression vector comprising the nucleotide
sequence of an
antibody or the nucleotide sequence of an antibody is transferred to a host
cell by conventional
techniques (e.g., electroporation, liposomal transfection, and calcium
phosphate precipitation),
and the transfected cells are then cultured by conventional techniques to
produce the antibody.
In specific embodiments, the expression of the antibody is regulated by a
constitutive, an
inducible or a tissue, specific promoter.
[0166] In some embodiments, a variety of host-expression vector systems is
utilized to express
an antibody or its binding fragment described herein. Such host-expression
systems represent
vehicles by which the coding sequences of the antibody is produced and
subsequently purified,
but also represent cells that are, when transformed or transfected with the
appropriate nucleotide
coding sequences, express an antibody or its binding fragment in situ. These
include, but are not
limited to, microorganisms such as bacteria (e.g., E. colt and B. subtilis)
transformed with
recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors
containing
an antibody or its binding fragment coding sequences; yeast (e.g.,
Saccharomyces Pichia)
transformed with recombinant yeast expression vectors containing an antibody
or its binding
fragment coding sequences; insect cell systems infected with recombinant virus
expression
vectors (e.g., baculovirus) containing an antibody or its binding fragment
coding sequences;
plant cell systems infected with recombinant virus expression vectors (e.g.,
cauliflower mosaic
virus (CaMV) and tobacco mosaic virus (TMV)) or transformed with recombinant
plasmid
expression vectors (e.g., Ti plasmid) containing an antibody or its binding
fragment coding
sequences; or mammalian cell systems (e.g., COS, CHO, BH, 293, 293T, 3T3
cells) harboring
recombinant expression constructs containing promoters derived from the genome
of
mammalian cells (e.g., metallothionein promoter) or from mammalian viruses
(e.g. the
adenovirus late promoter; the vaccinia virus 7.5K promoter).
[0167] For long-term, high-yield production of recombinant proteins, stable
expression is
preferred. In some instances, cell lines that stably express an antibody are
optionally engineered.
Rather than using expression vectors that contain viral origins of
replication, host cells are
transformed with DNA controlled by appropriate expression control elements
(e.g., promoter,
enhancer, sequences, transcription terminators, polyadenylation sites, etc.),
and a selectable
marker. Following the introduction of the foreign DNA, engineered cells are
then allowed to
grow for 1-2 days in an enriched media, and then are switched to a selective
media. The
selectable marker in the recombinant plasmid confers resistance to the
selection and allows cells
to stably integrate the plasmid into their chromosomes and grow to form foci
that in turn are
cloned and expanded into cell lines. This method can advantageously be used to
engineer cell
lines which express the antibody or its binding fragments.
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[0168] In some instances, a number of selection systems are used, including
but not limited to
the herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell 11:223),
hypoxanthine-
guanine phosphoribosyltransferase (Szybalska & Szybalski, 192, Proc. Natl.
Acad. Sci. USA
48:202), and adenine phosphoribosyltransferase (Lowy et al., 1980, Cell
22:817) genes are
employed in tk¨, hgprt¨ or aprt¨ cells, respectively. Also, antimetabolite
resistance are used as
the basis of selection for the following genes: dhfr, which confers resistance
to methotrexate
(Wigler et al., 1980, Proc. Natl. Acad. Sci. USA 77:357; O'Hare et al., 1981,
Proc. Natl. Acad.
Sci. USA 78:1527); gpt, which confers resistance to mycophenolic acid
(Mulligan & Berg, 1981,
Proc. Natl. Acad. Sci. USA 78:2072); neo, which confers resistance to the
aminoglycoside G-
418 (Clinical Pharmacy 12:488-505; Wu and Wu, 1991, Biotherapy 3:87-95;
Tolstoshev, 1993,
Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan, 1993, Science 260:926-932;
and Morgan
and Anderson, 1993, Ann. Rev. Biochem. 62:191-217; May, 1993, TIB TECH
11(5):155-215)
and hygro, which confers resistance to hygromycin (Santerre et al., 1984, Gene
30:147).
Methods commonly known in the art of recombinant DNA technology which can be
used are
described in Ausubel et al. (eds., 1993, Current Protocols in Molecular
Biology, John Wiley &
Sons, NY; Kriegler, 1990, Gene Transfer and Expression, A Laboratory Manual,
Stockton
Press, NY; and in Chapters 12 and 13, Dracopoli et al. (eds), 1994, Current
Protocols in Human
Genetics, John Wiley & Sons, NY.; Colberre-Garapin et al., 1981, 1. Mol. Biol.
150:1).
[0169] In some instances, the expression levels of an antibody are increased
by vector
amplification (for a review, see Bebbington and Hentschel, The use of vectors
based on gene
amplification for the expression of cloned genes in mammalian cells in DNA
cloning, Vol. 3.
(Academic Press, New York, 1987)). When a marker in the vector system
expressing an
antibody is amplifiable, an increase in the level of inhibitor present in
culture of host cell will
increase the number of copies of the marker gene. Since the amplified region
is associated with
the nucleotide sequence of the antibody, production of the antibody will also
increase (Crouse et
al., 1983, Mol. Cell Biol. 3:257).
[0170] In some instances, any method known in the art for purification of an
antibody is used,
for example, by chromatography (e.g., ion exchange, affinity, particularly by
affinity for the
specific antigen after Protein A, and sizing column chromatography),
centrifugation, differential
solubility, or by any other standard technique for the purification of
proteins.
Polymer Conjugating Moiety
[0171] In some embodiments, a polymer moiety C is further conjugated to a
polynucleic acid
molecule described herein, a binding moiety described herein, or in
combinations thereof In
some instances, a polymer moiety C is conjugated a polynucleic acid molecule.
In some cases, a
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polymer moiety C is conjugated to a binding moiety. In other cases, a polymer
moiety C is
conjugated to a polynucleic acid molecule-binding moiety molecule. In
additional cases, a
polymer moiety C is conjugated, and as discussed under the Therapeutic
Molecule Platform
section.
[0172] In some instances, the polymer moiety C is a natural or synthetic
polymer, consisting
of long chains of branched or unbranched monomers, and/or cross-linked network
of monomers
in two or three dimensions. In some instances, the polymer moiety C includes a
polysaccharide,
lignin, rubber, or polyalkylene oxide (e.g., polyethylene glycol). In some
instances, the at least
one polymer moiety C includes, but is not limited to, alpha-, omega-
dihydroxylpolyethyleneglycol, biodegradable lactone-based polymer, e.g.
polyacrylic acid,
polylactide acid (PLA), poly(glycolic acid) (PGA), polypropylene, polystyrene,
polyolefin,
polyamide, polycyanoacrylate, polyimide, polyethylenterephthalat (PET, PETG),
polyethylene
terephthalate (PETE), polytetramethylene glycol (PTG), or polyurethane as well
as mixtures
thereof As used herein, a mixture refers to the use of different polymers
within the same
compound as well as in reference to block copolymers. In some cases, block
copolymers are
polymers wherein at least one section of a polymer is build up from monomers
of another
polymer. In some instances, the polymer moiety C comprises polyalkylene oxide.
In some
instances, the polymer moiety C comprises PEG. In some instances, the polymer
moiety C
comprises polyethylene imide (PEI) or hydroxy ethyl starch (HES).
[0173] In some instances, C is a PEG moiety. In some instances, the PEG moiety
is
conjugated at the 5' terminus of the polynucleic acid molecule while the
binding moiety is
conjugated at the 3' terminus of the polynucleic acid molecule. In some
instances, the PEG
moiety is conjugated at the 3' terminus of the polynucleic acid molecule while
the binding
moiety is conjugated at the 5' terminus of the polynucleic acid molecule. In
some instances, the
PEG moiety is conjugated to an internal site of the polynucleic acid molecule.
In some
instances, the PEG moiety, the binding moiety, or a combination thereof, are
conjugated to an
internal site of the polynucleic acid molecule. In some instances, the
conjugation is a direct
conjugation. In some instances, the conjugation is via native ligation.
[0174] In some embodiments, the polyalkylene oxide (e.g., PEG) is a
polydispers or
monodispers compound. In some instances, polydispers material comprises
disperse distribution
of different molecular weight of the material, characterized by mean weight
(weight average)
size and dispersity. In some instances, the monodisperse PEG comprises one
size of molecules.
In some embodiments, C is poly- or monodispersed polyalkylene oxide (e.g.,
PEG) and the
indicated molecular weight represents an average of the molecular weight of
the polyalkylene
oxide, e.g., PEG, molecules.
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[0175] In some embodiments, the molecular weight of the polyalkylene oxide
(e.g., PEG) is
about 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400,
1450, 1500, 1600,
1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900,
3000, 3250,
3350, 3500, 3750, 4000, 4250, 4500, 4600, 4750, 5000, 5500, 6000, 6500, 7000,
7500, 8000,
10,000, 12,000, 20,000, 35,000, 40,000, 50,000, 60,000, or 100,000 Da.
[0176] In some embodiments, C is polyalkylene oxide (e.g., PEG) and has a
molecular weight
of about 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400,
1450, 1500,
1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800,
2900, 3000,
3250, 3350, 3500, 3750, 4000, 4250, 4500, 4600, 4750, 5000, 5500, 6000, 6500,
7000, 7500,
8000, 10,000, 12,000, 20,000, 35,000, 40,000, 50,000, 60,000, or 100,000 Da.
In some
embodiments, C is PEG and has a molecular weight of about 200, 300, 400, 500,
600, 700, 800,
900, 1000, 1100, 1200, 1300, 1400, 1450, 1500, 1600, 1700, 1800, 1900, 2000,
2100, 2200,
2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3250, 3350, 3500, 3750, 4000,
4250, 4500,
4600, 4750, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 10,000, 12,000, 20,000,
35,000, 40,000,
50,000, 60,000, or 100,000 Da. In some instances, the molecular weight of C is
about 200 Da.
In some instances, the molecular weight of C is about 300 Da. In some
instances, the molecular
weight of C is about 400 Da. In some instances, the molecular weight of C is
about 500 Da. In
some instances, the molecular weight of C is about 600 Da. In some instances,
the molecular
weight of C is about 700 Da. In some instances, the molecular weight of C is
about 800 Da. In
some instances, the molecular weight of C is about 900 Da. In some instances,
the molecular
weight of C is about 1000 Da. In some instances, the molecular weight of C is
about 1100 Da.
In some instances, the molecular weight of C is about 1200 Da. In some
instances, the molecular
weight of C is about 1300 Da. In some instances, the molecular weight of C is
about 1400 Da.
In some instances, the molecular weight of C is about 1450 Da. In some
instances, the
molecular weight of C is about 1500 Da. In some instances, the molecular
weight of C is about
1600 Da. In some instances, the molecular weight of C is about 1700 Da. In
some instances,
the molecular weight of C is about 1800 Da. In some instances, the molecular
weight of C is
about 1900 Da. In some instances, the molecular weight of C is about 2000 Da.
In some
instances, the molecular weight of C is about 2100 Da. In some instances, the
molecular weight
of C is about 2200 Da. In some instances, the molecular weight of C is about
2300 Da. In some
instances, the molecular weight of C is about 2400 Da. In some instances, the
molecular weight
of C is about 2500 Da. In some instances, the molecular weight of C is about
2600 Da. In some
instances, the molecular weight of C is about 2700 Da. In some instances, the
molecular weight
of C is about 2800 Da. In some instances, the molecular weight of C is about
2900 Da. In some
instances, the molecular weight of C is about 3000 Da. In some instances, the
molecular weight
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of C is about 3250 Da. In some instances, the molecular weight of C is about
3350 Da. In some
instances, the molecular weight of C is about 3500 Da. In some instances, the
molecular weight
of C is about 3750 Da. In some instances, the molecular weight of C is about
4000 Da. In some
instances, the molecular weight of C is about 4250 Da. In some instances, the
molecular weight
of C is about 4500 Da. In some instances, the molecular weight of C is about
4600 Da. In some
instances, the molecular weight of C is about 4750 Da. In some instances, the
molecular weight
of C is about 5000 Da. In some instances, the molecular weight of C is about
5500 Da. In some
instances, the molecular weight of C is about 6000 Da. In some instances, the
molecular weight
of C is about 6500 Da. In some instances, the molecular weight of C is about
7000 Da. In some
instances, the molecular weight of C is about 7500 Da. In some instances, the
molecular weight
of C is about 8000 Da. In some instances, the molecular weight of C is about
10,000 Da. In
some instances, the molecular weight of C is about 12,000 Da. In some
instances, the molecular
weight of C is about 20,000 Da. In some instances, the molecular weight of C
is about 35,000
Da. In some instances, the molecular weight of C is about 40,000 Da. In some
instances, the
molecular weight of C is about 50,000 Da. In some instances, the molecular
weight of C is
about 60,000 Da. In some instances, the molecular weight of C is about 100,000
Da.
[0177] In some embodiments, the polyalkylene oxide (e.g., PEG) is a discrete
PEG, in which
the discrete PEG is a polymeric PEG comprising more than one repeating
ethylene oxide units.
In some instances, a discrete PEG (dPEG) comprises from 2 to 60, from 2 to 50,
or from 2 to 48
repeating ethylene oxide units. In some instances, a dPEG comprises about 2,
3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26, 28, 30, 35, 40, 42,
48, 50 or more repeating
ethylene oxide units. In some instances, a dPEG comprises about 2 or more
repeating ethylene
oxide units. In some instances, a dPEG comprises about 3 or more repeating
ethylene oxide
units. In some instances, a dPEG comprises about 4 or more repeating ethylene
oxide units. In
some instances, a dPEG comprises about 5 or more repeating ethylene oxide
units. In some
instances, a dPEG comprises about 6 or more repeating ethylene oxide units. In
some instances,
a dPEG comprises about 7 or more repeating ethylene oxide units. In some
instances, a dPEG
comprises about 8 or more repeating ethylene oxide units. In some instances, a
dPEG comprises
about 9 or more repeating ethylene oxide units. In some instances, a dPEG
comprises about 10
or more repeating ethylene oxide units. In some instances, a dPEG comprises
about 11 or more
repeating ethylene oxide units. In some instances, a dPEG comprises about 12
or more repeating
ethylene oxide units. In some instances, a dPEG comprises about 13 or more
repeating ethylene
oxide units. In some instances, a dPEG comprises about 14 or more repeating
ethylene oxide
units. In some instances, a dPEG comprises about 15 or more repeating ethylene
oxide units. In
some instances, a dPEG comprises about 16 or more repeating ethylene oxide
units. In some
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instances, a dPEG comprises about 17 or more repeating ethylene oxide units.
In some instances,
a dPEG comprises about 18 or more repeating ethylene oxide units. In some
instances, a dPEG
comprises about 19 or more repeating ethylene oxide units. In some instances,
a dPEG
comprises about 20 or more repeating ethylene oxide units. In some instances,
a dPEG
comprises about 22 or more repeating ethylene oxide units. In some instances,
a dPEG
comprises about 24 or more repeating ethylene oxide units. In some instances,
a dPEG
comprises about 26 or more repeating ethylene oxide units. In some instances,
a dPEG
comprises about 28 or more repeating ethylene oxide units. In some instances,
a dPEG
comprises about 30 or more repeating ethylene oxide units. In some instances,
a dPEG
comprises about 35 or more repeating ethylene oxide units. In some instances,
a dPEG
comprises about 40 or more repeating ethylene oxide units. In some instances,
a dPEG
comprises about 42 or more repeating ethylene oxide units. In some instances,
a dPEG
comprises about 48 or more repeating ethylene oxide units. In some instances,
a dPEG
comprises about 50 or more repeating ethylene oxide units. In some cases, a
dPEG is
synthesized as a single molecular weight compound from pure (e.g., about 95%,
98%, 99%, or
99.5%) staring material in a step-wise fashion. In some cases, a dPEG has a
specific molecular
weight, rather than an average molecular weight. In some cases, a dPEG
described herein is a
dPEG from Quanta Biodesign, LMD.
[0178] In some embodiments, the polymer moiety C comprises a cationic mucic
acid-based
polymer (cMAP). In some instances, cMPA comprises one or more subunit of at
least one
repeating subunit, and the subunit structure is represented as Formula (III):
NH 2+ OH OH 0
H
NH2+ 111 H "n 0 OH OH H
Formula III
wherein m is independently at each occurrence 1, 2, 3, 4, 5, 6, 7, 8, 9, or
10, preferably 4-6 or 5;
and n is independently at each occurrence 1, 2, 3, 4, or 5. In some
embodiments, m and n are,
for example, about 10.
[0179] In some instances, cMAP is further conjugated to a PEG moiety,
generating a cMAP-
PEG copolymer, an mPEG-cMAP-PEGm triblock polymer, or a cMAP-PEG-cMAP triblock
polymer. In some instances, the PEG moiety is in a range of from about 500 Da
to about 50,000
Da. In some instances, the PEG moiety is in a range of from about 500 Da to
about 1000 Da,
greater than 1000 Da to about 5000 Da, greater than 5000 Da to about 10,000
Da, greater than
10,000 to about 25,000 Da, greater than 25,000 Da to about 50,000 Da,or any
combination of
two or more of these ranges.
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[0180] In some instances, the polymer moiety C is cMAP-PEG copolymer, an mPEG-
cMAP-
PEGm triblock polymer, or a cMAP-PEG-cMAP triblock polymer. In some cases, the
polymer
moiety C is cMAP-PEG copolymer. In other cases, the polymer moiety C is an
mPEG-cMAP-
PEGm triblock polymer. In additional cases, the polymer moiety C is a cMAP-PEG-
cMAP
triblock polymer.
Endosomolytic or Cell Membrane Penetration Moiety
[0181] In some embodiments, a molecule of Formula (Xa): A-X1-B'-X2-C, further
comprises
an additional conjugating moiety. In some instances, the additional
conjugating moiety is an
endosomolytic moiety and/or a cell membrane penetration moiety. In some cases,
the
endosomolytic moiety is a cellular compartmental release component, such as a
compound
capable of releasing from any of the cellular compartments known in the art,
such as the
endosome, lysosome, endoplasmic reticulum (ER), Golgi apparatus, microtubule,
peroxisome,
or other vesicular bodies with the cell. In some cases, the endosomolytic
moiety comprises an
endosomolytic polypeptide, an endosomolytic polymer, an endosomolytic lipid,
or an
endosomolytic small molecule. In some cases, the endosomolytic moiety
comprises an
endosomolytic polypeptide. In other cases, the endosomolytic moiety comprises
an
endosomolytic polymer. In some cases, the cell membrane penetration moiety
comprises a cell
penetrating peptide (CPP). In other cases, the cell membrane penetration
moiety comprises a cell
penetrating lipid. In other cases, the cell membrane penetration moiety
comprises a cell
penetrating small molecule.
Endosomolytic and Cell Membrane Penetration Polypeptides
[0182] In some embodiments, a molecule of Formula (Xa): A-X1-B-X2-C, is
further
conjugated with an endosomolytic polypeptide. In some cases, the endosomolytic
polypeptide is
a pH-dependent membrane active peptide. In some cases, the endosomolytic
polypeptide is an
amphipathic polypeptide. In additional cases, the endosomolytic polypeptide is
a
peptidomimetic. In some instances, the endosomolytic polypeptide comprises
INF, melittin,
meucin, or their respective derivatives thereof In some instances, the
endosomolytic
polypeptide comprises INF or its derivatives thereof In other cases, the
endosomolytic
polypeptide comprises melittin or its derivatives thereof In additional cases,
the endosomolytic
polypeptide comprises meucin or its derivatives thereof In some instances, the
endosomolytic
polypeptide comprises Pep-1 (originated from NLS from Simian Virus 40 large
antigen and
reverse transcriptase of HIV), Pvec (originated from VE-Cadherin), VT5
(originated from
synthetic peptide), C105Y(originated from 1-antitrypsin), transportan
(originated from Galanin
and mastoparan), TP10 (originated from Galanin and mastoparan), MPG
(originated from a
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hydrophobic domain from the fusion sequence of HIV gp41 and NLS of SV40 T
antigen),
GH625 (originated from glycoprotein gH of HSV type I), CADY (PPTG1 peptide),
GALA
(synthetic peptide), INF (Influenza HA2 fusion peptide), HA2E5-TAT (Influenza
HA2 subunit
of influenza virus X31 strain fusion peptide), HA2-penetratin (Influenza HA2
subunit of
influenza virus X31 strain fusion peptide), HA-K4 (Influenza HA2 subunit of
influenza virus
X31 strain fusion peptide), HA2E4 (Influenza HA2 subunit of influenza virus
X31 strain fusion
peptide), H5WYG (HA2 analogue), GALA-INF3-(PEG)6-NH (INF3 fusion peptide), or
CM18-
TAT11 (Cecropin-A-Melittin2_12 (CM18) fusion peptide).
[0183] In some cases, the endosomolytic moiety comprises a Bak BH3 polypeptide
which
induces apoptosis through antagonization of suppressor targets such as Bc1-2
and/or Bc1-xL. In
some instances, the endosomolytic moiety comprises a Bak BH3 polypeptide
described in
Albarran, et at., "Efficient intracellular delivery of a pro-apoptotic peptide
with a pH-responsive
carrier," Reactive & Functional Polymers 71: 261-265 (2011).
[0184] In some instances, the endosomolytic moiety comprises a polypeptide
(e.g., a cell-
penetrating polypeptide) as described in PCT Publication Nos. W02013/166155 or
W02015/069587.
Endosomolytic Lipids
[0185] In some embodiments, the endosomolytic moiety is a lipid (e.g., a
fusogenic lipid). In
some embodiments, a molecule of Formula (Xa): A-X1-B'- X2-C, is further
conjugated with an
endosomolytic lipid (e.g., fusogenic lipid). Exemplary fusogenic lipids
include 1,2-dileoyl-sn-3-
phosphoethanolamine (DOPE), phosphatidylethanolamine (POPE),
palmitoyloleoylphosphatidylcholine (POPC), (6Z, 9Z,28Z,31Z)-heptatriaconta-6,
9,28,31-tetraen-
19-ol (Di-Lin), N-methyl(2,2-di((9Z,12Z)-octadeca-9,12-dieny1)-1,3-dioxolan-4-
yl)methanamine (DLin-k-DMA) and N-methy1-2-(2,2-di((9Z,12Z)-octadeca-9,12-
dieny1)-1,3-
dioxolan-4-yl)ethanamine (XTC). In some instances, an endosomolytic moiety is
a lipid (e.g., a
fusogenic lipid) described in PCT Publication No. W009/126,933.
Endosomolytic Small Molecules
[0186] In some embodiments, the endosomolytic moiety is a small molecule. In
some
embodiments, a molecule of Formula (Xa): A-X1-B'- X2-C, is further conjugated
with an
endosomolytic small molecule. Exemplary small molecules suitable as
endosomolytic moieties
include, but are not limited to, quinine, chloroquine, hydroxychloroquines,
amodiaquins
(carnoquines), amopyroquines, primaquines, mefloquines, nivaquines,
halofantrines, quinone
imines, or a combination thereof In some instances, quinoline endosomolytic
moieties include,
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but are not limited to, 7-chloro-4-(4-diethylamino- 1 -methylbutyl-
amino)quinoline (chloroquine);
7-chloro-4-(4-ethyl-(2-hydroxyethyl)-amino- 1-methylbutyl-amino)quinoline
(hydroxychloroquine); 7-fluoro-4-(4-diethylamino - 1 -methylbutyl-
amino)quinoline; 4-(4-
diethylamino- 1 -methylbutylamino) quinoline; 7-hydroxy-4-(4-diethyl-amino- 1 -

methylbutylamino)quinoline; 7-chloro-4-(4-diethylamino- 1-butylamino)quinoline
(desmethylchloroquine); 7-fluoro-4-(4-diethylamino- 1-butylamino)quinoline); 4-
(4-diethyl-
amino-1 -butylamino)quinoline; 7-hydroxy-4-(4-diethylamino- 1-
butylamino)quinoline; 7-chloro-
4-(1-carboxy-4-diethylamino- 1 -butylamino)quinoline; 7-fluoro-4-(1-carboxy-4-
diethyl-amino- 1 -
butylamino)quinoline; 4-(1-carboxy-4-diethylamino- 1 -butylamino) quinoline; 7-
hydroxy-4-(1-
carboxy-4-diethylamino- 1 -butylamino)quinoline; 7-chloro-4-(1-carboxy-4-
diethylamino- 1 -
methylbutylamino)quinoline; 7-fluoro-4-(1 -carboxy-4-diethyl-amino- 1 -
methylbutylamino)quinoline; 4-(1-carboxy-4-diethylamino- 1 -
methylbutylamino)quinoline; 7-
hydroxy-4-(1 -carboxy-4-diethylamino- 1 -methylbutylamino)quinoline; 7-fluoro-
4-(4-ethyl-(2-
hydroxyethyl)-amino- 1 -methylbutylamino)quinoline; 4-(4-ethyl-(2-hydroxy-
ethyl)-amino- 1 -
methylbutylamino-)quinoline; 7-hydroxy-4-(4-ethyl-(2-hydroxyethyl)-amino- 1 -
methylbutylamino)quinoline; hydroxychloroquine phosphate; 7 -chloro-4-(4-ethyl-
(2-
hydroxyethyl- 1)-amino- 1 -butylamino)quinoline (desmethylhydroxychloroquine);
7 -fluoro-4-(4-
ethyl-(2-hydroxyethyl)-amino- 1 -butylamino)quinoline; 4-(4-ethyl-(2-
hydroxyethyl)-amino- 1 -
butylamino)quinoline; 7-hydroxy-4-(4-ethyl-(2-hydroxyethyl)-amino- 1 -
butylamino) quinoline;
7-chloro-4-(1 -carboxy-4-ethyl-(2-hydroxyethyl)-amino- 1 -
butylamino)quinoline; 7-fluoro-4-(1 -
carboxy-4-ethyl-(2-hydroxyethyl)-amino- 1 -butylamino)quinoline; 4-(1 -carboxy-
4-ethyl-(2-
hydroxyethyl)-amino- 1 -butylamino)quinoline; 7-hydroxy-4-(1 -carboxy-4-ethyl-
(2-
hydroxyethyl)-amino- 1 -butylamino)quinoline; 7-chloro-4-(1 -carboxy-4-ethyl-
(2-hydroxyethyl)-
amino- 1 -methylbutylamino)quinoline; 7-fluoro-4-(1 -carboxy-4-ethyl-(2-
hydroxyethyl)-amino- 1 -
methylbutylamino)quinoline; 4-(1-carboxy-4-ethyl -(2-hydroxyethyl)-amino- 1 -
methylbutylamino)quinoline; 7-hydroxy-4-(1 -carboxy-4-ethyl-(2-hydroxyethyl)-
amino- 1 -
methylbutylamino)quinoline; 8-[(4-aminopentyl)amino-6-methoxydihydrochloride
quinoline; 1 -
acetyl- 1,2,3 ,4-tetrahydroquinoline; 8- [(4-aminop entypamino] -6-
methoxyquinoline
dihydrochloride; 1 -butyryl- 1,2,3 ,4-tetrahydroquinoline; 3 -chloro-4-(4-
hydroxy-alpha,alpha'-
bis(2-methyl- 1 -pyrrolidiny1)-2, 5 -xylidinoquinoline, 4-[(4-diethyl-amino)-
1 -methylbutyl-amino] -
6-methoxyquinoline; 3 -fluoro-4-(4-hydroxy-alpha,alpha'-bis(2-methyl- 1 -
pyrrolidiny1)-2, 5 -
xylidinoquinoline, 4-[(4-diethylamino)- 1 -methylbutyl-amino] -6-
methoxyquinoline; 4-(4-
hydroxy-alpha,alpha'-bis(2-methyl- 1 -pyrrolidiny1)-2,5 -xylidinoquinoline; 4-
[(4-diethylamino)-
1 -methylbutyl-amino] -6-methoxyquinoline; 3 ,4-dihydro- 1 -(2H)-
quinolinecarboxyaldehyde;
1, 1 '-pentamethylene diquinoleinium diiodide; 8 -quinolinol sulfate and
amino, aldehyde,
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carboxylic, hydroxyl, halogen, keto, sulfhydryl and vinyl derivatives or
analogs thereof In
some instances, an endosomolytic moiety is a small molecule described in
Naisbitt et al (1997, J
Pharmacol Exp Therapy 280:884-893) and in U.S. Patent No. 5,736,557.
Cell Penetrating Polypeptide (CPP)
In some embodiments, cell penetrating polypeptide comprises positively charged
short peptides
with 5-30 amino acids. In some embodiments, cell penetrating polypeptide
comprises arginine
or lysine rich amino acid sequences. In some embodiments, cell penetrating
polypeptide
includes any polypeptide or combination thereof, including Antennapedia
Penetratin (43-58),
HIV-1 TAT protein (48-60), pVEC Cadherin (615-632), Transportan
Galanine/Mastoparan,
MPG HIV-gp41/5V40 T-antigen, Pep-1 HIV-reverse transcriptase/5V40 T-antigen,
Polyarginines, MAP, R6W3, NLS, 8-lysines, ARF (1-22), and Azurin-p28.
Linkers
[0187] In some embodiments, a linker described herein is a cleavable linker or
a non-cleavable
linker. In some instances, the linker is a cleavable linker. In some
instances, the linker is an
acid cleavable linker. In some instances, the linker is a non-cleavable
linker. In some instances,
the linker includes a Ci-C6 alkyl group (e.g., a C5, C4, C3, C2, or Ci alkyl
group). In some
instances, the linker includes homobifunctional cross linkers,
heterobifunctional cross linkers,
and the like. In some instances, the liker is a traceless linker (or a zero-
length linker). In some
instances, the linker is a non-polymeric linker. In some cases, the linker is
a non-peptide linker
or a linker that does not contain an amino acid residue.
[0188] In some instances, the linker comprises a homobifunctional linker.
Exemplary
homobifunctional linkers include, but are not limited to, Lomant's reagent
dithiobis
(succinimidylpropionate) DSP, 3'3'-dithiobis(sulfosuccinimidyl proprionate
(DTS SP),
disuccinimidyl suberate (D SS), bis(sulfosuccinimidyl)suberate (BS),
disuccinimidyl tartrate
(DST), disulfosuccinimidyl tartrate (sulfo DST), ethylene
glycobis(succinimidylsuccinate)
(EGS), disuccinimidyl glutarate (DSG), N,N'-disuccinimidyl carbonate (DSC),
dimethyl
adipimidate (DMA), dimethyl pimelimidate (DMP), dimethyl suberimidate (DMS),
dimethyl-
3,3 '-dithiobispropionimidate (DTBP), 1,4-di-3'-(2'-
pyridyldithio)propionamido)butane
(DPDPB), bismaleimidohexane (BMH), aryl halide-containing compound (DFDNB),
such as
e.g. 1,5-difluoro-2,4-dinitrobenzene or 1,3-difluoro-4,6-dinitrobenzene, 4,4'-
difluoro-3,3'-
dinitrophenylsulfone (DFDNPS), bis-[3-(4-azidosalicylamido)ethyl]disulfide
(BASED),
formaldehyde, glutaraldehyde, 1,4-butanediol diglycidyl ether, adipic acid
dihydrazide,
carbohydrazide, o-toluidine, 3,3'-dimethylbenzidine, benzidine, a,a'-p-
diaminodiphenyl, diiodo-
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p-xylene sulfonic acid, N,N'-ethylene-bis(iodoacetamide), or N,N'-
hexamethylene-
bis(iodoacetamide).
[0189] In some embodiments, the linker comprises a heterobifunctional linker.
Exemplary
heterobifunctional linker include, but are not limited to, amine-reactive and
sulfhydryl cross-
linkers such as N-succinimidyl 3-(2-pyridyldithio)propionate (sPDP), long-
chain N-
succinimidyl 3-(2-pyridyldithio)propionate (LC-sPDP), water-soluble-long-chain
N-
succinimidyl 3-(2-pyridyldithio) propionate (sulfo-LC-sPDP),
succinimidyloxycarbonyl-a-
methyl-a-(2-pyridyldithio)toluene (sMPT), sulfosuccinimidyl-6- a-methyl-a-(2-
pyridyldithio)toluamido]hexanoate (sulfo-LC-sMPT), succinimidy1-4-(N-
maleimidomethyl)cyclohexane-1-carboxylate (sMCC), sulfosuccinimidy1-4-(N-
maleimidomethyl)cyclohexane-1-carboxylate (sulfo-sMCC), m-maleimidobenzoyl-N-
hydroxysuccinimide ester (MB s), m-maleimidobenzoyl-N-hydroxysulfosuccinimide
ester
(sulfo-MB s), N-succinimidy1(4-iodoacteyl)aminobenzoate (sIAB),
sulfosuccinimidy1(4-
iodoacteyl)aminobenzoate (sulfo-sIAB), succinimidyl-4-(p-
maleimidophenyl)butyrate (sMPB),
sulfosuccinimidyl-4-(p-maleimidophenyl)butyrate (sulfo-sMPB), N-(y-
maleimidobutyryloxy)succinimide ester (GMBs), N-(y-
maleimidobutyryloxy)sulfosuccinimide
ester (sulfo-GMB s), succinimidyl 6-((iodoacetyl)amino)hexanoate (sIAX),
succinimidyl 646-
(((iodoacetyl)amino)hexanoyl)amino]hexanoate (sIAXX), succinimidyl 4-
(((iodoacetyl)amino)methyl)cyclohexane-1-carboxylate (sIAC), succinimidyl 6-
((((4-
iodoacetyl)amino)methyl)cyclohexane-1-carbonyl)amino) hexanoate (sIACX), p-
nitrophenyl
iodoacetate (NPIA), carbonyl-reactive and sulfhydryl-reactive cross-linkers
such as 4-(4-N-
maleimidophenyl)butyric acid hydrazide (MPBH), 4-(N-
maleimidomethyl)cyclohexane-1-
carboxyl-hydrazide-8 (M2C2H), 3-(2-pyridyldithio)propionyl hydrazide (PDPH),
amine-reactive
and photoreactive cross-linkers such as N-hydroxysuccinimidy1-4-azidosalicylic
acid (NHs-
AsA), N-hydroxysulfosuccinimidy1-4-azidosalicylic acid (sulfo-NHs-AsA),
sulfosuccinimidyl-
(4-azidosalicylamido)hexanoate (sulfo-NHs-LC-AsA), sulfosuccinimidy1-2-(p-
azidosalicylamido)ethy1-1,31-dithiopropionate (sAsD), N-hydroxysuccinimidy1-4-
azidobenzoate
(HsAB), N-hydroxysulfosuccinimidy1-4-azidobenzoate (sulfo-HsAB), N-
succinimidy1-6-(41-
azido-2'-nitrophenylamino)hexanoate (sANPAH), sulfosuccinimidy1-6-(41-azido-T-
nitrophenylamino)hexanoate (sulfo-sANPAH), N-5-azido-2-
nitrobenzoyloxysuccinimide (ANB-
NOs), sulfosuccinimidyl-2-(m-azido-o-nitrobenzamido)-ethyl-1,31-
dithiopropionate (sAND), N-
succinimidy1-4(4-azidopheny1)1,3 '-dithiopropionate (sADP), N-
sulfosuccinimidy1(4-
azidopheny1)-1,31-dithiopropionate (sulfo-sADP), sulfosuccinimidyl 4-(p-
azidophenyl)butyrate
(sulfo-sAPB), sulfosuccinimidyl 2-(7-azido-4-methylcoumarin-3-acetamide)ethy1-
1,3'-
dithiopropionate (sAED), sulfosuccinimidyl 7-azido-4-methylcoumain-3-acetate
(sulfo-
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sAMCA), p-nitrophenyl diazopyruvate (pNPDP), p-nitropheny1-2-diazo-3,3,3-
trifluoropropionate (PNP-DTP), sulfhydryl-reactive and photoreactive cross-
linkers such as1-(p-
Azidosalicylamido)-4-(iodoacetamido)butane (AsIB), N44-(p-
azidosalicylamido)buty1]-31-(2'-
pyridyldithio)propionamide (APDP), benzophenone-4-iodoacetamide, benzophenone-
4-
maleimide carbonyl-reactive and photoreactive cross-linkers such as p-
azidobenzoyl hydrazide
(ABH), carboxylate-reactive and photoreactive cross-linkers such as
azidosalicylamido)butylamine (AsBA), and arginine-reactive and photoreactive
cross-linkers
such as p-azidophenyl glyoxal (APG).
[0190] In some instances, the linker comprises a reactive functional group. In
some cases, the
reactive functional group comprises a nucleophilic group that is reactive to
an electrophilic
group present on a binding moiety. Exemplary electrophilic groups include
carbonyl groups¨
such as aldehyde, ketone, carboxylic acid, ester, amide, enone, acyl halide or
acid anhydride. In
some embodiments, the reactive functional group is aldehyde. Exemplary
nucleophilic groups
include hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazine
carboxylate, and
arylhydrazide.
[0191] In some embodiments, the linker comprises a maleimide group. In some
instances, the
maleimide group is also referred to as a maleimide spacer. In some instances,
the maleimide
group further encompasses a caproic acid, forming maleimidocaproyl (mc). In
some cases, the
linker comprises maleimidocaproyl (mc). In some cases, the linker is
maleimidocaproyl (mc).
In other instances, the maleimide group comprises a maleimidomethyl group,
such as
succinimidy1-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sMCC) or
sulfosuccinimidy1-
4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-sMCC) described above.
[0192] In some embodiments, the maleimide group is a self-stabilizing
maleimide. In some
instances, the self-stabilizing maleimide utilizes diaminopropionic acid (DPR)
to incorporate a
basic amino group adjacent to the maleimide to provide intramolecular
catalysis of
tiosuccinimide ring hydrolysis, thereby eliminating maleimide from undergoing
an elimination
reaction through a retro-Michael reaction. In some instances, the self-
stabilizing maleimide is a
maleimide group described in Lyon, et at., "Self-hydrolyzing maleimides
improve the stability
and pharmacological properties of antibody-drug conjugates," Nat. Biotechnol.
32(10):1059-
1062 (2014). In some instances, the linker comprises a self-stabilizing
maleimide. In some
instances, the linker is a self-stabilizing maleimide.
[0193] In some embodiments, the linker comprises a peptide moiety. In some
instances, the
peptide moiety comprises at least 2, 3, 4, 5, 6, 7, 8, or more amino acid
residues. In some
instances, the peptide moiety is a cleavable peptide moiety (e.g., either
enzymatically or
chemically). In some instances, the peptide moiety is a non-cleavable peptide
moiety. In some
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instances, the peptide moiety comprises Val-Cit (valine-citrulline), Gly-Gly-
Phe-Gly (SEQ ID
NO: 14223), Phe-Lys, Val-Lys, Gly-Phe-Lys, Phe-Phe-Lys, Ala-Lys, Val-Arg, Phe-
Cit, Phe-
Arg, Leu-Cit, Ile-Cit, Trp-Cit, Phe-Ala, Ala-Leu-Ala-Leu (SEQ ID NO: 14224),
or Gly-Phe-
Leu-Gly (SEQ ID NO: 14225). In some instances, the linker comprises a peptide
moiety such
as: Val-Cit (valine-citrulline), Gly-Gly-Phe-Gly (SEQ ID NO: 14223), Phe-Lys,
Val-Lys, Gly-
Phe-Lys, Phe-Phe-Lys, Ala-Lys, Val-Arg, Phe-Cit, Phe-Arg, Leu-Cit, Ile-Cit,
Trp-Cit, Phe-Ala,
Ala-Leu-Ala-Leu (SEQ ID NO: 14224), or Gly-Phe-Leu-Gly (SEQ ID NO: 14225). In
some
cases, the linker comprises Val-Cit. In some cases, the linker is Val-Cit.
[0194] In some embodiments, the linker comprises a benzoic acid group, or its
derivatives
thereof In some instances, the benzoic acid group or its derivatives thereof
comprise
paraaminobenzoic acid (PABA). In some instances, the benzoic acid group or its
derivatives
thereof comprise gamma-aminobutyric acid (GABA).
[0195] In some embodiments, the linker comprises one or more of a maleimide
group, a
peptide moiety, and/or a benzoic acid group, in any combination. In some
embodiments, the
linker comprises a combination of a maleimide group, a peptide moiety, and/or
a benzoic acid
group. In some instances, the maleimide group is maleimidocaproyl (mc). In
some instances,
the peptide group is val-cit. In some instances, the benzoic acid group is
PABA. In some
instances, the linker comprises a mc-val-cit group. In some cases, the linker
comprises a val-cit-
PABA group. In additional cases, the linker comprises a mc-val-cit-PABA group.
[0196] In some embodiments, the linker is a self-immolative linker or a self-
elimination
linker. In some cases, the linker is a self-immolative linker. In other cases,
the linker is a self-
elimination linker (e.g., a cyclization self-elimination linker). In some
instances, the linker
comprises a linker described in U.S. Patent No. 9,089,614 or PCT Publication
No.
W02015038426.
[0197] In some embodiments, the linker is a dendritic type linker. In some
instances, the
dendritic type linker comprises a branching, multifunctional linker moiety. In
some instances,
the dendritic type linker is used to increase the molar ratio of
polynucleotide B to the binding
moiety A. In some instances, the dendritic type linker comprises PAMAM
dendrimers.
[0198] In some embodiments, the linker is a traceless linker or a linker in
which after cleavage
does not leave behind a linker moiety (e.g., an atom or a linker group) to a
binding moiety A, a
polynucleotide B, a polymer C, or an endosomolytic moiety D. Exemplary
traceless linkers
include, but are not limited to, germanium linkers, silicium linkers, sulfur
linkers, selenium
linkers, nitrogen linkers, phosphorus linkers, boron linkers, chromium
linkers, or
phenylhydrazide linker. In some cases, the linker is a traceless aryl-triazene
linker as described
in Hejesen, et at., "A traceless aryl-triazene linker for DNA-directed
chemistry," Org Biomol
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Chem 11(15): 2493-2497 (2013). In some instances, the linker is a traceless
linker described in
Blaney, et at., "Traceless solid-phase organic synthesis," Chem. Rev. 102:
2607-2024 (2002). In
some instances, a linker is a traceless linker as described in U.S. Patent No.
6,821,783.
[0199] In some instances, the linker comprises a functional group that exerts
steric hinderance
at the site of bonding between the linker and a conjugating moiety (e.g., A,
B, C, or D described
herein). In some instances, the steric hinderance is a steric hindrance around
a disulfide bond.
Exemplary linkers that exhibit steric hinderance comprises a
heterobifunctional linker, such as a
heterobifunctional linker described above. In some cases, a linker that
exhibits steric hinderance
comprises SMCC and SPDB.
[0200] In some instances, the linker is an acid cleavable linker. In some
instances, the acid
cleavable linker comprises a hydrazone linkage, which is susceptible to
hydrolytic cleavage. In
some cases, the acid cleavable linker comprises a thiomaleamic acid linker. In
some cases, the
acid cleavable linker is a thiomaleamic acid linker as described in Castaneda,
et at, "Acid-
cleavable thiomaleamic acid linker for homogeneous antibody-drug conjugation,"
Chem.
Commun. 49: 8187-8189 (2013).
[0201] In some instances, the linker is a linker described in U.S. Patent Nos.
6,884,869;
7,498,298; 8,288,352; 8,609,105; or 8,697,688; U.S. Patent Publication Nos.
2014/0127239;
2013/028919; 2014/286970; 2013/0309256; 2015/037360; or 2014/0294851; or PCT
Publication Nos. W02015057699; W02014080251; W02014197854; W02014145090; or
W02014177042.
[0202] In some embodiments, X, Y, and L are independently a bond or a linker.
In some
instances, X, Y, and L are independently a bond. In some cases, X, Y, and L
are independently
a linker.
[0203] In some instances, X is a bond or a linker. In some instances, X is a
bond. In some
instances, X is a linker. In some instances, the linker is a Ci-C6 alkyl
group. In some cases, X is
a Ci-C6 alkyl group, such as for example, a C5, C4, C3, C2, or Ci alkyl group.
In some cases, the
Ci-C6 alkyl group is an unsubstituted Ci-C6 alkyl group. As used in the
context of a linker, and
in particular in the context of X, alkyl means a saturated straight or
branched hydrocarbon
radical containing up to six carbon atoms. In some instances, X is a non-
polymeric linker. In
some instances, X includes a homobifunctional linker or a heterobifunctional
linker described
supra. In some cases, X includes a heterobifunctional linker. In some cases, X
includes sMCC.
In other instances, X includes a heterobifunctional linker optionally
conjugated to a C1-C6 alkyl
group. In other instances, X includes sMCC optionally conjugated to a Ci-C6
alkyl group. In
additional instances, X does not include a homobifunctional linker or a
heterobifunctional linker
described supra.
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[0204] In some instances, Y is a bond or a linker. In some instances, Y is a
bond. In other
cases, Y is a linker. In some embodiments, Y is a Ci-C6 alkyl group. In some
instances, Y is a
homobifunctional linker or a heterobifunctional linker described supra. In
some instances, Y is
a homobifunctional linker described supra. In some instances, Y is a
heterobifunctional linker
described supra. In some instances, Y comprises a maleimide group, such as
maleimidocaproyl
(mc) or a self-stabilizing maleimide group described above. In some instances,
Y comprises a
peptide moiety, such as Val-Cit. In some instances, Y comprises a benzoic acid
group, such as
PABA. In additional instances, Y comprises a combination of a maleimide group,
a peptide
moiety, and/or a benzoic acid group. In additional instances, Y comprises a mc
group. In
additional instances, Y comprises a mc-val-cit group. In additional instances,
Y comprises a
val-cit-PABA group. In additional instances, Y comprises a mc-val-cit-PABA
group.
[0205] In some instances, L is a bond or a linker. In some cases, L is a bond.
In other cases,
L is a linker. In some embodiments, L is a Ci-C6 alkyl group. In some
instances, L is a
homobifunctional linker or a heterobifunctional linker described supra. In
some instances, L is
a homobifunctional linker described supra. In some instances, L is a
heterobifunctional linker
described supra. In some instances, L comprises a maleimide group, such as
maleimidocaproyl
(mc) or a self-stabilizing maleimide group described above. In some instances,
L comprises a
peptide moiety, such as Val-Cit. In some instances, L comprises a benzoic acid
group, such as
PABA. In additional instances, L comprises a combination of a maleimide group,
a peptide
moiety, and/or a benzoic acid group. In additional instances, L comprises a mc
group. In
additional instances, L comprises a mc-val-cit group. In additional instances,
L comprises a val-
cit-PABA group. In additional instances, L comprises a mc-val-cit-PABA group.
Methods of Use
[0206] In some embodiments, a composition or a pharmaceutical formulation
described herein
comprising a binding moiety conjugated to a polynucleic acid molecule and a
polymer is used
for the treatment of a disease or disorder. In some instances, the disease or
disorder is a muscle
dystrophy, muscle atrophy, and/or muscle wasting. Muscle dystrophy refers to a
loss of muscle
mass and/or to a progressive weakening and degeneration of muscles. In some
cases, the loss of
muscle mass and/or the progressive weakening and degeneration of muscles
occurs due to a high
rate of protein degradation, a low rate of protein synthesis, or a combination
of both. In some
cases, a high rate of muscle protein degradation is due to muscle protein
catabolism (i.e., the
breakdown of muscle protein in order to use amino acids as substrates for
gluconeogenesis). In
some instances, the disease or disorder is a cancer. In some embodiments, a
composition or a
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pharmaceutical formulation described herein is used as an immunotherapy for
the treatment of a
disease or disorder. In some instances, the immunotherapy is an immuno-
oncology therapy.
Cancer
[0207] In some embodiments, a composition or a pharmaceutical formulation
described herein
is used for the treatment of cancer. In some instances, the cancer is a solid
tumor. In some
instances, the cancer is a hematologic malignancy. In some instances, the
cancer is a relapsed or
refractory cancer, or a metastatic cancer. In some instances, the solid tumor
is a relapsed or
refractory solid tumor, or a metastatic solid tumor. In some cases, the
hematologic malignancy
is a relapsed or refractory hematologic malignancy, or a metastatic
hematologic malignancy.
[0208] In some embodiments, the cancer is a solid tumor. In some instances, a
composition or
a pharmaceutical formulation described herein comprising an oligonucleotide,
optionally
conjugated to a binding moiety, a polymer, or a combination thereof is used
for the treatment of
a solid tumor. In some instances, a composition or a pharmaceutical
formulation described
herein comprising an oligonucleotide, optionally conjugated to a binding
moiety, a polymer, or a
combination thereof is used for the treatment of anal cancer, appendix cancer,
bile duct cancer
(i.e., cholangiocarcinoma), bladder cancer, brain tumor, breast cancer,
cervical cancer, colon
cancer, cancer of Unknown Primary (CUP), esophageal cancer, eye cancer,
fallopian tube
cancer, gastroenterological cancer, kidney cancer, liver cancer, lung cancer,
medulloblastoma,
melanoma, oral cancer, ovarian cancer, pancreatic cancer, parathyroid disease,
penile cancer,
pituitary tumor, prostate cancer, rectal cancer, skin cancer, stomach cancer,
testicular cancer,
throat cancer, thyroid cancer, uterine cancer, vaginal cancer, or vulvar
cancer. In some
instances, the solid tumor is a relapsed or refractory solid tumor, or a
metastatic solid tumor.
[0209] In some instances, the cancer is a hematologic malignancy. In some
instances, a
composition or a pharmaceutical formulation described herein comprising an
oligonucleotide,
optionally conjugated to a binding moiety, a polymer, or a combination thereof
is used for the
treatment of a hematologic malignancy. In some instances, a composition or a
pharmaceutical
formulation described herein comprising an oligonucleotide, optionally
conjugated to a binding
moiety, a polymer, or a combination thereof is used for the treatment of a
leukemia, a
lymphoma, a myeloma, a non-Hodgkin's lymphoma, or a Hodgkin's lymphoma. In
some
instances, the hematologic malignancy comprises chronic lymphocytic leukemia
(CLL), small
lymphocytic lymphoma (SLL), high risk CLL, a non-CLL/SLL lymphoma,
prolymphocytic
leukemia (PLL), follicular lymphoma (FL), diffuse large B-cell lymphoma
(DLBCL), mantle
cell lymphoma (MCL), Waldenstrom's macroglobulinemia, multiple myeloma,
extranodal
marginal zone B cell lymphoma, nodal marginal zone B cell lymphoma, Burkitt's
lymphoma,
non-Burkitt high grade B cell lymphoma, primary mediastinal B-cell lymphoma
(PMBL),
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immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, B cell
prolymphocytic leukemia, lymphoplasmacytic lymphoma, splenic marginal zone
lymphoma,
plasma cell myeloma, plasmacytoma, mediastinal (thymic) large B cell lymphoma,
intravascular
large B cell lymphoma, primary effusion lymphoma, or lymphomatoid
granulomatosis. In some
cases, the hematologic malignancy is a relapsed or refractory hematologic
malignancy, or a
metastatic hematologic malignancy.
[0210] In some instances, the cancer is a KRAS-associated, EGFR-associated, AR-
associated
cancer, HPRT1-associated cancer, or 13-catenin associated cancer. In some
instances, a
composition or a pharmaceutical formulation described herein comprising an
oligonucleotide,
optionally conjugated to a binding moiety, a polymer, or a combination thereof
is used for the
treatment of a KRAS-associated, EGFR-associated, AR-associated cancer, HPRT1-
associated
cancer, or 13-catenin associated cancer. In some instances, a composition or a
pharmaceutical
formulation described herein comprising an oligonucleotide, optionally
conjugated to a binding
moiety, a polymer, or a combination thereof is used for the treatment of a
KRAS-associated
cancer. In some instances, a composition or a pharmaceutical formulation
described herein
comprising an oligonucleotide, optionally conjugated to a binding moiety, a
polymer, or a
combination thereof is used for the treatment of an EGFR-associated cancer. In
some instances,
a composition or a pharmaceutical formulation described herein comprising an
oligonucleotide,
optionally conjugated to a binding moiety, a polymer, or a combination thereof
is used for the
treatment of an AR-associated cancer. In some instances, a composition or a
pharmaceutical
formulation described herein comprising an oligonucleotide, optionally
conjugated to a binding
moiety, a polymer, or a combination thereof is used for the treatment of an
HPRT1-associated
cancer. In some instances, a composition or a pharmaceutical formulation
described herein
comprising an oligonucleotide, optionally conjugated to a binding moiety, a
polymer, or a
combination thereof is used for the treatment of a 13-catenin associated
cancer. In some
instances, the cancer is a solid tumor. In some instances, the cancer is a
hematologic
malignancy. In some instances, the solid tumor is a relapsed or refractory
solid tumor, or a
metastatic solid tumor. In some cases, the hematologic malignancy is a
relapsed or refractory
hematologic malignancy, or a metastatic hematologic malignancy. In some
instances, the cancer
comprises bladder cancer, breast cancer, colorectal cancer, endometrial
cancer, esophageal
cancer, glioblastoma multiforme, head and neck cancer, kidney cancer, lung
cancer, ovarian
cancer, pancreatic cancer, prostate cancer, thyroid cancer, acute myeloid
leukemia, CLL,
DLBCL, or multiple myeloma. In some instances, the 13-catenin associated
cancer further
comprises PIK3C-associated cancer and/or MYC-associated cancer.
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Immunotherapy
[0211] In some embodiments, a composition or a pharmaceutical formulation
described herein
is used as an immunotherapy for the treatment of a disease or disorder. In
some instances, the
immunotherapy is an immuno-oncology therapy. In some instances, immuno-
oncology therapy
is categorized into active, passive, or combinatory (active and passive)
methods. In active
immuno-oncology therapy method, for example, tumor-associated antigens (TAAs)
are
presented to the immune system to trigger an attack on cancer cells presenting
these TAAs. In
some instances, the active immune-oncology therapy method includes tumor-
targeting and/or
immune-targeting agents (e.g., checkpoint inhibitor agents such as monoclonal
antibodies),
and/or vaccines, such as in situ vaccination and/or cell-based or non-cell
based (e.g., dendritic
cell-based, tumor cell-based, antigen, anti-idiotype, DNA, or vector-based)
vaccines. In some
instances, the cell-based vaccines are vaccines which are generated using
activated immune cells
obtained from a patient's own immune system which are then activated by the
patient's own
cancer. In some instances, the active immune-oncology therapy is further
subdivided into non-
specific active immunotherapy and specific active immunotherapy. In some
instances, non-
specific active immunotherapy utilizes cytokines and/or other cell signaling
components to
induce a general immune system response. In some cases, specific active
immunotherapy
utilizes specific TAAs to elicit an immune response.
[0212] In some embodiments, a composition or a pharmaceutical formulation
described herein
is used as an active immuno-oncology therapy method for the treatment of a
disease or disorder
(e.g., cancer). In some embodiments, the composition or a pharmaceutical
formulation
described herein comprises a tumor-targeting agent. In some instances, the
tumor-targeting
agent is encompassed by a binding moiety A. In other instances, the tumor-
targeting agent is an
additional agent used in combination with a molecule of Formula (I). In some
instances, the
tumor-targeting agent is a tumor-directed polypeptide (e.g., a tumor-directed
antibody). In some
instances, the tumor-targeting agent is a tumor-directed antibody, which
exerts its antitumor
activity through mechanisms such as direct killing (e.g., signaling-induced
apoptosis),
complement-dependent cytotoxicity (CDC), and/or antibody-dependent cell-
mediated
cytotoxicity (ADCC). In additional instances, the tumor-targeting agent
elicits an adaptive
immune response, with the induction of antitumor T cells.
[0213] In some embodiments, the binding moiety A is a tumor-directed
polypeptide (e.g., a
tumor-directed antibody). In some instances, the binding moiety A is a tumor-
directed antibody,
which exerts its antitumor activity through mechanisms such as direct killing
(e.g., signaling-
induced apoptosis), complement-dependent cytotoxicity (CDC), and/or antibody-
dependent cell-
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mediated cytotoxicity (ADCC). In additional instances, the binding moiety A
elicits an adaptive
immune response, with the induction of antitumor T cells.
[0214] In some embodiments, the composition or a pharmaceutical formulation
described
herein comprises an immune-targeting agent. In some instances, the immune-
targeting agent is
encompassed by a binding moiety A. In other instances, the immune-targeting
agent is an
additional agent used in combination with a molecule of Formula (I). In some
instances, the
immune-targeting agent comprises cytokines, checkpoint inhibitors, or a
combination thereof
[0215] In some embodiments, the immune-targeting agent is a checkpoint
inhibitor. In some
cases, an immune checkpoint molecule is a molecule presented on the cell
surface of CD4
and/or CD8 T cells. Exemplary immune checkpoint molecules include, but are not
limited to,
Programmed Death-Ligand 1 (PD-L1, also known as B7-H1, CD274), Programmed
Death 1
(PD-1), CTLA-4, B7H1, B7H4, OX- 40, CD137, CD40, 2B4, ID01, ID02, VISTA, CD27,
CD28, PD-L2 (B7-DC, CD273), LAG3, CD80, CD86, PDL2, B7H3, HVEM, BTLA, KIR,
GAL9, TIM3, A2aR, MARCO (macrophage receptor with collageneous structure), PS
(phosphatidylserine), ICOS (inducible T cell costimulator), HAVCR2, CD276,
VTCN1, CD70,
and CD160.
[0216] In some instances, an immune checkpoint inhibitor refers to any
molecule that
modulates or inhibits the activity of an immune checkpoint molecule. In some
instances,
immune checkpoint inhibitors include antibodies, antibody-derivatives (e.g.,
Fab fragments,
scFvs, minobodies, diabodies), antisense oligonucleotides, siRNA, aptamers, or
peptides. In
some embodiments, an immune checkpoint inhibitor is an inhibitor of Programmed
Death-
Ligand 1 (PD-L1, also known as B7-H1, CD274), Programmed Death 1 (PD-1), CTLA-
4, PD-
L2 (B7-DC, CD273), LAG3, TIM3, 2B4, A2aR, B7H1, B7H3, B7H4, BTLA, CD2, CD27,
CD28, CD30, CD40, CD70, CD80, CD86, CD137,CD160, CD226, CD276, DR3, GAL9,
GITR,
HAVCR2, HVEM, ID01, ID02, ICOS (inducible T cell costimulator), KIR, LAIR1,
LIGHT,
MARCO (macrophage receptor with collageneous structure), PS
(phosphatidylserine), OX- 40,
SLAM, TIGHT, VISTA, VTCN1, or any combinations thereof
[0217] In some embodiments, exemplary checkpoint inhibitors include:
[0218] PD-Li inhibitors such as Genentech's MPDL3280A (RG7446), Anti-mouse PD-
Li
antibody Clone 10F.9G2 (Cat # BE0101) from BioXcell, anti-PD-Li monoclonal
antibody
M1DX-1105 (BMS-936559) and BMS-935559 from Bristol-Meyer's Squibb,
MSB0010718C,
mouse anti-PD-Li Clone 29E.2A3, and AstraZeneca's MEDI4736;
[0219] PD-L2 inhibitors such as GlaxoSmithKline's AMP-224 (Amplimmune), and
rHIgMl2B7;
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[0220] PD-1 inhibitors such as anti-mouse PD-1 antibody Clone J43 (Cat #
BE0033-2) from
BioXcell, anti-mouse PD-1 antibody Clone RMP1-14 (Cat # BE0146) from BioXcell,
mouse
anti-PD-1 antibody Clone EH12, Merck's MK-3475 anti-mouse PD-1 antibody
(Keytruda,
pembrolizumab, lambrolizumab), AnaptysBio's anti-PD-1 antibody known as
ANB011,
antibody MDX-1 106 (ONO-4538), Bristol-Myers Squibb's human IgG4 monoclonal
antibody
nivolumab (Opdivog, BMS-936558, MDX1106), AstraZeneca's AMP-514 and AMP-224,
and
Pidilizumab (CT-011) from CureTech Ltd;
[0221] CTLA-4 inhibitors such as Bristol Meyers Squibb's anti-CTLA-4 antibody
ipilimumab
(also known as Yervoyg, MDX-010, BMS-734016 and MDX-101), anti-CTLA4 Antibody,
clone 9H10 from Millipore, Pfizer's tremelimumab (CP-675,206, ticilimumab),
and anti-CTLA4
antibody clone BNI3 from Abcam;
[0222] LAG3 inhibitors such as anti-Lag-3 antibody clone eBioC9B7W (C9B7W)
from
eBioscience, anti-Lag3 antibody LS-B2237 from LifeSpan Biosciences, IMP321
(ImmuFact)
from Immutep, anti-Lag3 antibody BMS-986016, and the LAG-3 chimeric antibody
A9H12;
[0223] B7-H3 inhibitors such as MGA271;
[0224] KIR inhibitors such as Lirilumab (IPH2101);
[0225] CD137 (41BB) inhibitors such as urelumab (BMS-663513, Bristol-Myers
Squibb), PF-
05082566 (anti-4-1BB, PF-2566, Pfizer), or XmAb-5592 (Xencor);
[0226] PS inhibitors such as Bavituximab; and inhibitors such as an antibody
or fragments
(e.g., a monoclonal antibody, a human, humanized, or chimeric antibody)
thereof, RNAi
molecules, or small molecules to TIM3, CD52, CD30, CD20, CD33, CD27, 0X40
(CD134),
GITR, ICOS, BTLA (CD272), CD160, 2B4, LAIR1, TIGHT, LIGHT, DR3, CD226, CD2, or
SLAM.
[0227] In some embodiments, a binding moiety A comprising an immune checkpoint
inhibitor
is used for the treatment of a disease or disorder (e.g., cancer). In some
instances, the binding
moiety A is a bispecific antibody or a binding fragment thereof that comprises
an immune
checkpoint inhibitor. In some cases, a binding moiety A comprising an
inhibitor of Programmed
Death-Ligand 1 (PD-L1, also known as B7-H1, CD274), Programmed Death 1 (PD-1),
CTLA-4,
PD-L2 (B7-DC, CD273), LAG3, TIM3, 2B4, A2aR, B7H1, B7H3, B7H4, BTLA, CD2,
CD27,
CD28, CD30, CD40, CD70, CD80, CD86, CD137,CD160, CD226, CD276, DR3, GAL9,
GITR,
HAVCR2, HVEM, ID01, ID02, ICOS (inducible T cell costimulator), KIR, LAIR1,
LIGHT,
MARCO (macrophage receptor with collageneous structure), PS
(phosphatidylserine), OX- 40,
SLAM, TIGHT, VISTA, VTCN1, or any combinations thereof, is used for the
treatment of a
disease or disorder (e.g., cancer).
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[0228] In some embodiments, a molecule of Formula (I) in combination with an
immune
checkpoint inhibitor is used for the treatment of a disease or disorder (e.g.,
cancer). In some
instances, the immune checkpoint inhibitor comprises an inhibitor of
Programmed Death-Ligand
1 (PD-L1, also known as B7-H1, CD274), Programmed Death 1 (PD-1), CTLA-4, PD-
L2 (B7-
DC, CD273), LAG3, TIM3, 2B4, A2aR, B7H1, B7H3, B7H4, BTLA, CD2, CD27, CD28,
CD30, CD40, CD70, CD80, CD86, CD137,CD160, CD226, CD276, DR3, GAL9, GITR,
HAVCR2, HVEM, IDOL ID02, ICOS (inducible T cell costimulator), KIR, LAIRL
LIGHT,
MARCO (macrophage receptor with collageneous structure), PS
(phosphatidylserine), OX- 40,
SLAM, TIGHT, VISTA, VTCN1, or any combinations thereof In some cases, a
molecule of
Formula (I) is used in combination with ipilimumab, tremelimumab, nivolumab,
pemrolizumab,
pidilizumab, MPDL3280A, MEDI4736, M5B0010718C, MK-3475, or BMS-936559, for the
treatment of a disease or disorder (e.g., cancer).
[0229] In some embodiments, the immune-targeting agent is a cytokine. In some
cases,
cytokine is further subgrouped into chemokine, interferon, interleukin, and
tumor necrosis
factor. In some embodiments, chemokine plays a role as a chemoattractant to
guide the
migration of cells, and is classified into four subfamilies: CXC, CC, CX3C,
and XC. Exemplary
chemokines include chemokines from the CC subfamily: CCL1, CCL2 (racp-i),
CCL3, CCL4,
CCL5 (RANTES), CCL6, CCL7, CCL8, CCL9 (or CCL10), CCL11, CCL12, CCL13, CCL14,
CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25,
CCL26, CCL27, and CCL28; the CXC subfamily: CXCL1, CXCL2, CXCL3, CXCL4, CXCL5,
CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14,
CXCL15, CXCL16, and CXCL17; the XC subfamily: XCL1 and XCL2; and the CX3C
subfamily CX3CL1.
[0230] Interferon (IFNs) comprises interferon type I (e.g., 1FN-a, IFN-e;
IFN-K, and
IFN-w), interferon type II (e.g. IFN-7), and interferon type III. In some
embodiments, IFN-a is
further classified into about 13 subtypes which include IFNAL IFNA2, IFNA4,
IFNA5, IFNA6,
IFNA7, IFNA8, IFNA10, IFNA13, IFNA142 IFNA162 IFNA172 and IFNA21.
[0231] Interleukin is expressed by leukocyte or white blood cell and promote
the development
and differentiation of T and B lymphocytes and hematopoietic cells. Exemplary
interleukins
include 1L-1, IL-2; 1L-3, IL-4, IL-5, IL-6; 1L-7, 1L-8 (CXCL8), 1L-9, IL-10,
IL-11; IL-12, IL-13,
1L-14, IL-15, IL-16,11,17, IL-18, IL-19,111,20, IL-21, 1L-22, IL-23, IL-
24,11,25, IL-26, IL-27,
1L-28, FL-29, 1L-30, IL-31, 1L-32, IL-33, IL-35, and IL-36.
[0232] Tumor necrosis factors (TNFs) are a group of cytokines that modulate
apoptosis. In
some instances, there are about 19 members within the TNT' family, including,
not limited to,
TNFa., lymphotoxin-alpha (LT-alpha), lymphotoxin-beta (LT-beta), T cell
antigen gp39
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(CD40L), CD27L, CD30L, FASL, 4-11313Lõ OX4OL, and TNT-related apoptosis
inducing ligand
(TRAIL).
[0233] In some embodiments, a molecule of Formula (I) in combination with a
cytokine is
used for the treatment of a disease or disorder (e.g., cancer). In some cases,
a molecule of
Formula (I) in combination with a chemokine is used for the treatment of a
disease or disorder
(e.g., cancer). In some cases, a molecule of Formula (I) in combination with
an interferon is
used for the treatment of a disease or disorder (e.g., cancer). In some cases,
a molecule of
Formula (I) in combination with an interleukin is used for the treatment of a
disease or disorder
(e.g., cancer). In some cases, a molecule of Formula (I) in combination with a
tumor necrosis
factor is used for the treatment of a disease or disorder (e.g., cancer). In
some instances, a
molecule of Formula (I) in combination with IL-10, IL-2, IL-7, IL-8, IL-15,
MCP-1 (CCL2),
MIP-la, RANTES, MCP-3, MIP5, CCL19, CCL21, CXCL2, CXCL9, CXCL10, or CXCL11 is
used for the treatment of a disease or disorder (e.g., cancer).
[0234] In some embodiments, the composition or a pharmaceutical formulation
described
herein comprises a vaccine. In some instances, the vaccine is an in situ
vaccination. In some
instances, the vaccine is a cell-based vaccine. In some instances, the vaccine
is a non-cell based
vaccine. In some instances, a molecule of Formula (I) in combination with
dendritic cell-based
vaccine is used for the treatment of a disease or disorder (e.g., cancer). In
some instances, a
molecule of Formula (I) in combination with tumor cell-based vaccine is used
for the treatment
of a disease or disorder (e.g., cancer). In some instances, a molecule of
Formula (I) in
combination with antigen vaccine is used for the treatment of a disease or
disorder (e.g., cancer).
In some instances, a molecule of Formula (I) in combination with anti-idiotype
vaccine is used
for the treatment of a disease or disorder (e.g., cancer). In some instances,
a molecule of
Formula (I) in combination with DNA vaccine is used for the treatment of a
disease or disorder
(e.g., cancer). In some instances, a molecule of Formula (I) in combination
with vector-based
vaccine is used for the treatment of a disease or disorder (e.g., cancer).
[0235] In some embodiments, a composition or a pharmaceutical formulation
described herein
is used as a passive immuno-oncology therapy method for the treatment of a
disease or disorder
(e.g., cancer). The passive method, in some instances, utilizes adoptive
immune system
components such as T cells, natural killer (NK) T cells, and/or chimeric
antigen receptor (CAR)
T cells generated exogenously to attack cancer cells.
[0236] In some embodiments, a molecule of Formula (I) in combination with a T-
cell based
therapeutic agent is used for the treatment of a disease or disorder (e.g.,
cancer). In some cases,
the T-cell based therapeutic agent is an activated T-cell agent that
recognizes one or more of a
CD cell surface marker described above. In some instances, the T-cell based
therapeutic agent
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comprises an activated T-cell agent that recognizes one or more of CD2, CD3,
CD4, CD5, CD8,
CD27, CD28, CD80, CD134, CD137, CD152, CD154, CD160, CD200R, CD223, CD226,
CD244, CD258, CD267, CD272, CD274, CD278, CD279, or CD357. In some instances,
a
molecule of Formula (I) in combination with an activated T-cell agent
recognizing one or more
of CD2, CD3, CD4, CD5, CD8, CD27, CD28, CD80, CD134, CD137, CD152, CD154,
CD160,
CD200R, CD223, CD226, CD244, CD258, CD267, CD272, CD274, CD278, CD279, or
CD357
is used for the treatment of a disease or disorder (e.g., cancer).
[0237] In some embodiments, a molecule of Formula (I) in combination with
natural killer
(NK) T cell-based therapeutic agent is used for the treatment of a disease or
disorder (e.g.,
cancer). In some instances, the NK-based therapeutic agent is an activated NK
agent that
recognizes one or more of a CD cell surface marker described above. In some
cases, the NK-
based therapeutic agent is an activated NK agent that recognizes one or more
of CD2, CD11 a,
CD11b, CD16, CD56, CD58, CD62L, CD85j, CD158a/b, CD158c, CD158e/f/k, CD158h/j,
CD159a, CD162, CD226, CD314, CD335, CD337, CD244, or CD319. In some instances,
a
molecule of Formula (I) in combination with an activated NK agent recognizing
one or more of
CD2, CD11a, CD11b, CD16, CD56, CD58, CD62L, CD85j, CD158a/b, CD158c,
CD158e/f/k,
CD158h/j, CD159a, CD162, CD226, CD314, CD335, CD337, CD244, or CD319 is used
for the
treatment of a disease or disorder (e.g., cancer).
[0238] In some embodiments, a molecule of Formula (I) in combination with CAR-
T cell-
based therapeutic agent is used for the treatment of a disease or disorder
(e.g., cancer).
[0239] In some embodiments, a molecule of Formula (I) in combination with an
additional
agent that destabilizes the endosomal membrane (or disrupts the endosomal-
lysosomal
membrane trafficking) is used for the treatment of a disease or disorder
(e.g., cancer). In some
instances, the additional agent comprises an antimitotic agent. Exemplary
antimitotic agents
include, but are not limited to, taxanes such as paclitaxel and docetaxel;
vinca alkaloids such as
vinblastine, vincristine, vindesine, and vinorelbine; cabazitaxel; colchicine;
eribulin;
estramustine; etoposide; ixabepilone; podophyllotoxin; teniposide; or
griseofulvin. In some
instances, the additional agent comprises paclitaxel, docetaxel, vinblastine,
vincristine,
vindesine, vinorelbine, cabazitaxel, colchicine, eribulin, estramustine,
etoposide, ixabepilone,
podophyllotoxin, teniposide, or griseofulvin. In some instances, the
additional agent comprises
taxol. In some instances, the additional agent comprises paclitaxel. In some
instances, the
additional agent comprises etoposide. In other instances, the additional agent
comprises vitamin
K3.
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[0240] In some embodiments, a composition or a pharmaceutical formulation
described herein
is used as a combinatory method (including for both active and passive
methods) in the
treatment of a disease or disorder (e.g., cancer).
Muscle Dystrophy, Muscle Atrophy, Muscle Wasting
[0241] In one embodiment, muscle dystrophy refers to a significant loss in
muscle strength.
By significant loss in muscle strength is meant a reduction of strength in
diseased, injured, or
unused muscle tissue in a subject relative to the same muscle tissue in a
control subject. In an
embodiment, a significant loss in muscle strength is a reduction in strength
of at least 10%, at
least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least
40%, at least 45%, at
least 50%, or more relative to the same muscle tissue in a control subject. In
another
embodiment, by significant loss in muscle strength is meant a reduction of
strength in unused
muscle tissue relative to the muscle strength of the same muscle tissue in the
same subject prior
to a period of nonuse. In an embodiment, a significant loss in muscle strength
is a reduction of at
least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least
35%, at least 40%, at
least 45%, at least 50%, or more relative to the muscle strength of the same
muscle tissue in the
same subject prior to a period of nonuse.
[0242] In another embodiment, muscle dystrophy refers to a significant loss in
muscle mass.
By significant loss in muscle mass is meant a reduction of muscle volume in
diseased, injured,
or unused muscle tissue in a subject relative to the same muscle tissue in a
control subject. In an
embodiment, a significant loss of muscle volume is at least 10%, at least 15%,
at least 20%, at
least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least
50%, or more relative to
the same muscle tissue in a control subject. In another embodiment, by
significant loss in muscle
mass is meant a reduction of muscle volume in unused muscle tissue relative to
the muscle
volume of the same muscle tissue in the same subject prior to a period of
nonuse. In an
embodiment, a significant loss in muscle tissue is at least 10%, at least 15%,
at least 20%, at
least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least
50%, or more relative to
the muscle volume of the same muscle tissue in the same subject prior to a
period of nonuse.
Muscle volume is optionally measured by evaluating the cross-section area of a
muscle such as
by Magnetic Resonance Imaging (e.g., by a muscle volume/cross-section area
(CSA) MRI
method).
[0243] Myotonic dystrophy is a multisystemic neuromuscular disease comprising
two main
types: myotonic dystrophy type 1 (DM1) and myotonic dystrophy type 2 (DM2).
DM1 is caused
by a dominantly inherited "CTG" repeat expansion in the gene DM protein kinase
(DMPK),
which when transcribed into mRNA, forms hairpins that bind with high affinity
to the
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Muscleblind-like (MBNL) family of proteins. MBNL proteins are involved in post-
transcriptional splicing and polyadenylatin site regulation and loss of the
MBNL protein
functions lead to downstream accumulation of nuclear foci and increase in mis-
splicing events
and subsequently to myotonia and other clinical symptoms.
[0244] In some embodiments, described herein is a method of treating muscle
dystrophy,
muscle atrophy, and/or muscle wasting in a subject, which comprises providing
a polynucleic
acid molecule described herein or a polynucleic acid molecule conjugate
described herein and
administering to the subject a therapeutically effective amount of the
polynucleic acid molecule
or polynucleic acid molecule conjugate to the subject in need thereof to treat
the muscular
dystrophy muscle atrophy, and/or muscle wasting. In some embodiments, the
polynucleic acid
molecules or polynucleic acid molecule conjugates target a gene transcripts
that are mutated or
upregulated such that downregulation, deletion, exon skipping of transcripts
are desired to treat
the diseases. In some embodiments, the polynucleic acid molecules or
polynucleic acid molecule
conjugates target DMPK mRNA, DMD mRNA, or GYS1 mRNA.
Pharmaceutical Formulation
[0245] In some embodiments, the pharmaceutical formulations described herein
are
administered to a subject by multiple administration routes, including but not
limited to,
parenteral (e.g., intravenous, subcutaneous, intramuscular), oral, intranasal,
buccal, rectal, or
transdermal administration routes. In some instances, the pharmaceutical
composition describe
herein is formulated for parenteral (e.g., intravenous, subcutaneous,
intramuscular)
administration. In other instances, the pharmaceutical composition describe
herein is formulated
for oral administration. In still other instances, the pharmaceutical
composition describe herein
is formulated for intranasal administration.
[0246] In some embodiments, the pharmaceutical formulations include, but are
not limited to,
aqueous liquid dispersions, self-emulsifying dispersions, solid solutions,
liposomal dispersions,
aerosols, solid dosage forms, powders, immediate-release formulations,
controlled-release
formulations, fast melt formulations, tablets, capsules, pills, delayed
release formulations,
extended release formulations, pulsatile release formulations,
multiparticulate formulations (e.g.,
nanoparticle formulations), and mixed immediate and controlled release
formulations.
[0247] In some instances, the pharmaceutical formulation includes
multiparticulate
formulations. In some instances, the pharmaceutical formulation includes
nanoparticle
formulations. In some instances, nanoparticles comprise cMAP, cyclodextrin, or
lipids. In
some cases, nanoparticles comprise solid lipid nanoparticles, polymeric
nanoparticles, self-
emulsifying nanoparticles, liposomes, microemulsions, or micellar solutions.
Additional
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exemplary nanoparticles include, but are not limited to, paramagnetic
nanoparticles,
superparamagnetic nanoparticles, metal nanoparticles, fullerene-like
materials, inorganic
nanotubes, dendrimers (such as with covalently attached metal chelates),
nanofibers, nanohorns,
nano-onions, nanorods, nanoropes and quantum dots. In some instances, a
nanoparticle is a
metal nanoparticle, e.g., a nanoparticle of scandium, titanium, vanadium,
chromium, manganese,
iron, cobalt, nickel, copper, zinc, yttrium, zirconium, niobium, molybdenum,
ruthenium,
rhodium, palladium, silver, cadmium, hafnium, tantalum, tungsten, rhenium,
osmium, iridium,
platinum, gold, gadolinium, aluminum, gallium, indium, tin, thallium, lead,
bismuth,
magnesium, calcium, strontium, barium, lithium, sodium, potassium, boron,
silicon, phosphorus,
germanium, arsenic, antimony, and combinations, alloys or oxides thereof.
[0248] In some instances, a nanoparticle includes a core or a core and a
shell, as in a core-shell
nanoparticle.
[0249] In some instances, a nanoparticle is further coated with molecules for
attachment of
functional elements (e.g., with one or more of a polynucleic acid molecule or
binding moiety
described herein). In some instances, a coating comprises chondroitin sulfate,
dextran sulfate,
carboxymethyl dextran, alginic acid, pectin, carragheenan, fucoidan,
agaropectin, porphyran,
karaya gum, gellan gum, xanthan gum, hyaluronic acids, glucosamine,
galactosamine, chitin (or
chitosan), polyglutamic acid, polyaspartic acid, lysozyme, cytochrome C,
ribonuclease,
trypsinogen, chymotrypsinogen, a-chymotrypsin, polylysine, polyarginine,
histone, protamine,
ovalbumin, dextrin, or cyclodextrin. In some instances, a nanoparticle
comprises a graphene-
coated nanoparticle.
[0250] In some cases, a nanoparticle has at least one dimension of less than
about 500nm,
400nm, 300nm, 200nm, or 100nm.
[0251] In some instances, the nanoparticle formulation comprises paramagnetic
nanoparticles,
superparamagnetic nanoparticles, metal nanoparticles, fullerene-like
materials, inorganic
nanotubes, dendrimers (such as with covalently attached metal chelates),
nanofibers, nanohorns,
nano-onions, nanorods, nanoropes or quantum dots. In some instances, a
polynucleic acid
molecule or a binding moiety described herein is conjugated either directly or
indirectly to the
nanoparticle. In some instances, at least 1, 5, 10, 15, 20, 30, 40, 50, 60,
70, 80, 90, 100, or more
polynucleic acid molecules or binding moieties described herein are conjugated
either directly or
indirectly to a nanoparticle.
[0252] In some embodiments, the pharmaceutical formulations include a carrier
or carrier
materials selected on the basis of compatibility with the composition
disclosed herein, and the
release profile properties of the desired dosage form. Exemplary carrier
materials include, e.g.,
binders, suspending agents, disintegration agents, filling agents,
surfactants, solubilizers,
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stabilizers, lubricants, wetting agents, diluents, and the like.
Pharmaceutically compatible
carrier materials include, but are not limited to, acacia, gelatin, colloidal
silicon dioxide, calcium
glycerophosphate, calcium lactate, maltodextrin, glycerine, magnesium
silicate,
polyvinylpyrrollidone (PVP), cholesterol, cholesterol esters, sodium
caseinate, soy lecithin,
taurocholic acid, phosphotidylcholine, sodium chloride, tricalcium phosphate,
dipotassium
phosphate, cellulose and cellulose conjugates, sugars sodium stearoyl
lactylate, carrageenan,
monoglyceride, diglyceride, pregelatinized starch, and the like. See, e.g.,
Remington: The
Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing
Company,
1995); Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing
Co., Easton,
Pennsylvania 1975; Liberman, H.A. and Lachman, L., Eds., Pharmaceutical Dosage
Forms,
Marcel Decker, New York, N.Y., 1980; and Pharmaceutical Dosage Forms and Drug
Delivery
Systems, Seventh Ed. (Lippincott Williams & Wilkins1999).
[0253] In some instances, the pharmaceutical formulations further include pH-
adjusting agents
or buffering agents which include acids such as acetic, boric, citric, lactic,
phosphoric and
hydrochloric acids; bases such as sodium hydroxide, sodium phosphate, sodium
borate, sodium
citrate, sodium acetate, sodium lactate and tris-hydroxymethylaminomethane;
and buffers such
as citrate/dextrose, sodium bicarbonate and ammonium chloride. Such acids,
bases and buffers
are included in an amount required to maintain pH of the composition in an
acceptable range.
[0254] In some instances, the pharmaceutical formulation includes one or more
salts in an
amount required to bring osmolality of the composition into an acceptable
range. Such salts
include those having sodium, potassium or ammonium cations and chloride,
citrate, ascorbate,
borate, phosphate, bicarbonate, sulfate, thiosulfate or bisulfite anions;
suitable salts include
sodium chloride, potassium chloride, sodium thiosulfate, sodium bisulfite and
ammonium
sulfate.
[0255] In some instances, the pharmaceutical formulations further include
diluent which are
used to stabilize compounds because they can provide a more stable
environment. Salts
dissolved in buffered solutions (which also can provide pH control or
maintenance) are utilized
as diluents in the art, including, but not limited to a phosphate buffered
saline solution. In
certain instances, diluents increase bulk of the composition to facilitate
compression or create
sufficient bulk for homogenous blend for capsule filling. Such compounds can
include e.g.,
lactose, starch, mannitol, sorbitol, dextrose, microcrystalline cellulose such
as Avicel ; dibasic
calcium phosphate, dicalcium phosphate dihydrate; tricalcium phosphate,
calcium phosphate;
anhydrous lactose, spray-dried lactose; pregelatinized starch, compressible
sugar, such as Di-
Pac (Amstar); mannitol, hydroxypropylmethylcellulose,
hydroxypropylmethylcellulose acetate
stearate, sucrose-based diluents, confectioner's sugar; monobasic calcium
sulfate monohydrate,
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calcium sulfate dihydrate; calcium lactate trihydrate, dextrates; hydrolyzed
cereal solids,
amylose; powdered cellulose, calcium carbonate; glycine, kaolin; mannitol,
sodium chloride;
inositol, bentonite, and the like.
[0256] In some cases, the pharmaceutical formulations include disintegration
agents or
disintegrants to facilitate the breakup or disintegration of a substance. The
term "disintegrate"
include both the dissolution and dispersion of the dosage form when contacted
with
gastrointestinal fluid. Examples of disintegration agents include a starch,
e.g., a natural starch
such as corn starch or potato starch, a pregelatinized starch such as National
1551 or Amij el , or
sodium starch glycolate such as Promogel or Explotab , a cellulose such as a
wood product,
methylcrystalline cellulose, e.g., Avicel , Avicel PH101, AvicerPH102, Avicel
PH105,
Elcema P100, Emcocel , Vivacel , Ming Tia , and SolkaFloc , methylcellulose,
croscarmellose, or a cross-linked cellulose, such as cross-linked sodium
carboxymethylcellulose
(Ac-Di- Sol ), cross-linked carboxymethylcellulose, or cross-linked
croscarmello se, a cross-
linked starch such as sodium starch glycolate, a cross-linked polymer such as
crospovidone, a
cross-linked polyvinylpyrrolidone, alginate such as alginic acid or a salt of
alginic acid such as
sodium alginate, a clay such as Veegum HV (magnesium aluminum silicate), a
gum such as
agar, guar, locust bean, Karaya, pectin, or tragacanth, sodium starch
glycolate, bentonite, a
natural sponge, a surfactant, a resin such as a cation-exchange resin, citrus
pulp, sodium lauryl
sulfate, sodium lauryl sulfate in combination starch, and the like.
[0257] In some instances, the pharmaceutical formulations include filling
agents such as
lactose, calcium carbonate, calcium phosphate, dibasic calcium phosphate,
calcium sulfate,
microcrystalline cellulose, cellulose powder, dextrose, dextrates, dextran,
starches,
pregelatinized starch, sucrose, xylitol, lactitol, mannitol, sorbitol, sodium
chloride, polyethylene
glycol, and the like.
[0258] Lubricants and glidants are also optionally included in the
pharmaceutical formulations
described herein for preventing, reducing or inhibiting adhesion or friction
of materials.
Exemplary lubricants include, e.g., stearic acid, calcium hydroxide, talc,
sodium stearyl
fumerate, a hydrocarbon such as mineral oil, or hydrogenated vegetable oil
such as
hydrogenated soybean oil (Sterotex ), higher fatty acids and their alkali-
metal and alkaline earth
metal salts, such as aluminum, calcium, magnesium, zinc, stearic acid, sodium
stearates,
glycerol, talc, waxes, Stearowet , boric acid, sodium benzoate, sodium
acetate, sodium chloride,
leucine, a polyethylene glycol (e.g., PEG-4000) or a methoxypolyethylene
glycol such as
CarbowaxTM, sodium oleate, sodium benzoate, glyceryl behenate, polyethylene
glycol,
magnesium or sodium lauryl sulfate, colloidal silica such as SyloidTM, CabOSil
, a starch such
as corn starch, silicone oil, a surfactant, and the like.
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[0259] Plasticizers include compounds used to soften the microencapsulation
material or film
coatings to make them less brittle. Suitable plasticizers include, e.g.,
polyethylene glycols such
as PEG 300, PEG 400, PEG 600, PEG 1450, PEG 3350, and PEG 800, stearic acid,
propylene
glycol, oleic acid, triethyl cellulose and triacetin. Plasticizers can also
function as dispersing
agents or wetting agents.
[0260] Solubilizers include compounds such as triacetin, triethylcitrate,
ethyl oleate, ethyl
caprylate, sodium lauryl sulfate, sodium doccusate, vitamin E TPGS,
dimethylacetamide, N-
methylpyrrolidone, N-hydroxyethylpyrrolidone, polyvinylpyrrolidone,
hydroxypropylmethyl
cellulose, hydroxypropyl cyclodextrins, ethanol, n-butanol, isopropyl alcohol,
cholesterol, bile
salts, polyethylene glycol 200-600, glycofurol, transcutol, propylene glycol,
dimethyl isosorbide,
and the like.
[0261] Stabilizers include compounds such as any antioxidation agents,
buffers, acids,
preservatives and the like.
[0262] Suspending agents include compounds such as polyvinylpyrrolidone, e.g.,
polyvinylpyrrolidone K12, polyvinylpyrrolidone K17, polyvinylpyrrolidone K25,
or
polyvinylpyrrolidone K30, vinyl pyrrolidone/vinyl acetate copolymer (S630),
polyethylene
glycol, e.g., the polyethylene glycol can have a molecular weight of about 300
to about 6000, or
about 3350 to about 4000, or about 7000 to about 5400, sodium
carboxymethylcellulose,
methylcellulose, hydroxypropylmethylcellulose, hydroxymethylcellulose acetate
stearate,
polysorbate-80, hydroxyethylcellulose, sodium alginate, gums, such as, e.g.,
gum tragacanth and
gum acacia, guar gum, xanthans, including xanthan gum, sugars, cellulosics,
such as, e.g.,
sodium carboxymethylcellulose, methylcellulo se, sodium carboxymethylcellulo
se,
hydroxypropylmethylcellulose, hydroxyethylcellulose, polysorbate-80, sodium
alginate,
polyethoxylated sorbitan monolaurate, polyethoxylated sorbitan monolaurate,
povidone and the
like.
[0263] Surfactants include compounds such as sodium lauryl sulfate, sodium
docusate, Tween
60 or 80, triacetin, vitamin E TPGS, sorbitan monooleate, polyoxyethylene
sorbitan monooleate,
polysorbates, polaxomers, bile salts, glyceryl monostearate, copolymers of
ethylene oxide and
propylene oxide, e.g., Pluronic (BASF), and the like. Additional surfactants
include
polyoxyethylene fatty acid glycerides and vegetable oils, e.g.,
polyoxyethylene (60)
hydrogenated castor oil; and polyoxyethylene alkylethers and alkylphenyl
ethers, e.g., octoxynol
10, octoxynol 40. Sometimes, surfactants is included to enhance physical
stability or for other
purposes.
[0264] Viscosity enhancing agents include, e.g., methyl cellulose, xanthan
gum,
carboxymethyl cellulose, hydroxypropyl cellulose, hydroxypropylmethyl
cellulose,
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hydroxypropylmethyl cellulose acetate stearate, hydroxypropylmethyl cellulose
phthalate,
carbomer, polyvinyl alcohol, alginates, acacia, chitosans and combinations
thereof
[0265] Wetting agents include compounds such as oleic acid, glyceryl
monostearate, sorbitan
monooleate, sorbitan monolaurate, triethanolamine oleate, polyoxyethylene
sorbitan
monooleate, polyoxyethylene sorbitan monolaurate, sodium docusate, sodium
oleate, sodium
lauryl sulfate, sodium doccusate, triacetin, Tween 80, vitamin E TPGS,
ammonium salts and the
like.
Therapeutic Regimens
[0266] In some embodiments, the pharmaceutical compositions described herein
are
administered for therapeutic applications. In some embodiments, the
pharmaceutical
composition is administered once per day, twice per day, three times per day
or more. The
pharmaceutical composition is administered daily, every day, every alternate
day, five days a
week, once a week, every other week, two weeks per month, three weeks per
month, once a
month, twice a month, three times per month, or more. The pharmaceutical
composition is
administered for at least 1 month, 2 months, 3 months, 4 months, 5 months, 6
months, 7 months,
8 months, 9 months, 10 months, 11 months, 12 months, 18 months, 2 years, 3
years, or more.
[0267] In some embodiments, one or more pharmaceutical compositions are
administered
simultaneously, sequentially, or at an interval period of time. In some
embodiments, one or
more pharmaceutical compositions are administered simultaneously. In some
cases, one or
more pharmaceutical compositions are administered sequentially. In additional
cases, one or
more pharmaceutical compositions are administered at an interval period of
time (e.g., the first
administration of a first pharmaceutical composition is on day one followed by
an interval of at
least 1, 2, 3, 4, 5, or more days prior to the administration of at least a
second pharmaceutical
composition).
[0268] In some embodiments, two or more different pharmaceutical compositions
are
coadministered. In some instances, the two or more different pharmaceutical
compositions are
coadministered simultaneously. In some cases, the two or more different
pharmaceutical
compositions are coadministered sequentially without a gap of time between
administrations. In
other cases, the two or more different pharmaceutical compositions are
coadministered
sequentially with a gap of about 0.5 hour, 1 hour, 2 hour, 3 hour, 12 hours, 1
day, 2 days, or
more between administrations.
[0269] In the case wherein the patient's status does improve, upon the
doctor's discretion the
administration of the composition is given continuously; alternatively, the
dose of the
composition being administered is temporarily reduced or temporarily suspended
for a certain
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length of time (i.e., a "drug holiday"). In some instances, the length of the
drug holiday varies
between 2 days and 1 year, including by way of example only, 2 days, 3 days, 4
days, 5 days, 6
days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, 35 days, 50 days,
70 days, 100 days,
120 days, 150 days, 180 days, 200 days, 250 days, 280 days, 300 days, 320
days, 350 days, or
365 days. The dose reduction during a drug holiday is from 10%400%, including,
by way of
example only, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,
75%,
80%, 85%, 90%, 95%, or 100%.
[0270] Once improvement of the patient's conditions has occurred, a
maintenance dose is
administered if necessary. Subsequently, the dosage or the frequency of
administration, or both,
are optionally reduced, as a function of the symptoms, to a level at which the
improved disease,
disorder or condition is retained.
[0271] In some embodiments, the amount of a given agent that correspond to
such an amount
varies depending upon factors such as the particular compound, the severity of
the disease, the
identity (e.g., weight) of the subject or host in need of treatment, but
nevertheless is routinely
determined in a manner known in the art according to the particular
circumstances surrounding
the case, including, e.g., the specific agent being administered, the route of
administration, and
the subject or host being treated. In some instances, the desired dose is
conveniently presented in
a single dose or as divided doses administered simultaneously (or over a short
period of time) or
at appropriate intervals, for example as two, three, four or more sub-doses
per day.
[0272] The foregoing ranges are merely suggestive, as the number of variables
in regard to an
individual treatment regime is large, and considerable excursions from these
recommended
values are not uncommon. Such dosages are altered depending on a number of
variables, not
limited to the activity of the compound used, the disease or condition to be
treated, the mode of
administration, the requirements of the individual subject, the severity of
the disease or
condition being treated, and the judgment of the practitioner.
[0273] In some embodiments, toxicity and therapeutic efficacy of such
therapeutic regimens
are determined by standard pharmaceutical procedures in cell cultures or
experimental animals,
including, but not limited to, the determination of the LD50 (the dose lethal
to 50% of the
population) and the ED50 (the dose therapeutically effective in 50% of the
population). The
dose ratio between the toxic and therapeutic effects is the therapeutic index
and it is expressed as
the ratio between LD50 and EDS . Compounds exhibiting high therapeutic indices
are
preferred. The data obtained from cell culture assays and animal studies are
used in formulating
a range of dosage for use in human. The dosage of such compounds lies
preferably within a
range of circulating concentrations that include the ED50 with minimal
toxicity. The dosage
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varies within this range depending upon the dosage form employed and the route
of
administration utilized.
Kits/Article of Manufacture
[0274] Disclosed herein, in certain embodiments, are kits and articles of
manufacture for use
with one or more of the compositions and methods described herein. Such kits
include a carrier,
package, or container that is compartmentalized to receive one or more
containers such as vials,
tubes, and the like, each of the container(s) comprising one of the separate
elements to be used
in a method described herein. Suitable containers include, for example,
bottles, vials, syringes,
and test tubes. In one embodiment, the containers are formed from a variety of
materials such as
glass or plastic.
[0275] The articles of manufacture provided herein contain packaging
materials. Examples of
pharmaceutical packaging materials include, but are not limited to, blister
packs, bottles, tubes,
bags, containers, bottles, and any packaging material suitable for a selected
formulation and
intended mode of administration and treatment.
[0276] For example, the container(s) include a molecule of Formula (Xa): A-X-
B'-Y-C, as
disclosed herein. Such kits optionally include an identifying description or
label or instructions
relating to its use in the methods described herein.
[0277] A kit typically includes labels listing contents and/or instructions
for use and package
inserts with instructions for use. A set of instructions will also typically
be included.
[0278] In one embodiment, a label is on or associated with the container. In
one embodiment,
a label is on a container when letters, numbers, or other characters forming
the label are
attached, molded or etched into the container itself; a label is associated
with a container when it
is present within a receptacle or carrier that also holds the container, e.g.,
as a package insert. In
one embodiment, a label is used to indicate that the contents are to be used
for a specific
therapeutic application. The label also indicates directions for use of the
contents, such as in the
methods described herein.
[0279] In certain embodiments, the pharmaceutical compositions are presented
in a pack or
dispenser device which contains one or more unit dosage forms containing a
compound
provided herein. The pack, for example, contains metal or plastic foil, such
as a blister pack. In
one embodiment, the pack or dispenser device is accompanied by instructions
for
administration. In one embodiment, the pack or dispenser is also accompanied
with a notice
associated with the container in form prescribed by a governmental agency
regulating the
manufacture, use, or sale of pharmaceuticals, which notice is reflective of
approval by the
agency of the form of the drug for human or veterinary administration. Such
notice, for example,
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is the labeling approved by the U.S. Food and Drug Administration for
prescription drugs, or the
approved product insert. In one embodiment, compositions containing a compound
provided
herein formulated in a compatible pharmaceutical carrier are also prepared,
placed in an
appropriate container, and labeled for treatment of an indicated condition.
Certain Terminology
[0280] Unless defined otherwise, all technical and scientific terms used
herein have the same
meaning as is commonly understood by one of skill in the art to which the
claimed subject
matter belongs. It is to be understood that the foregoing general description
and the following
detailed description are exemplary and explanatory only and are not
restrictive of any subject
matter claimed. In this application, the use of the singular includes the
plural unless specifically
stated otherwise. It must be noted that, as used in the specification and the
appended claims, the
singular forms "a," "an" and "the" include plural referents unless the context
clearly dictates
otherwise. In this application, the use of "or" means "and/or" unless stated
otherwise.
Furthermore, use of the term "including" as well as other forms, such as
"include", "includes,"
and "included," is not limiting.
[0281] As used herein, ranges and amounts can be expressed as "about" a
particular value or
range. About also includes the exact amount. Hence "about 5 l.L" means "about
5 l.L" and also
"5 L." Generally, the term "about" includes an amount that is expected to be
within
experimental error.
[0282] The section headings used herein are for organizational purposes only
and are not to be
construed as limiting the subject matter described.
[0283] As used herein, the terms "individual(s)", "subject(s)" and
"patient(s)" mean any
mammal. In some embodiments, the mammal is a human. In some embodiments, the
mammal is
a non-human. None of the terms require or are limited to situations
characterized by the
supervision (e.g. constant or intermittent) of a health care worker (e.g. a
doctor, a registered
nurse, a nurse practitioner, a physician's assistant, an orderly or a hospice
worker).
Chemical Definitions
[0284] The abbreviations used herein have their conventional meaning within
the chemical
and biological arts. The chemical structures and formulae set forth herein are
constructed
according to the standard rules of chemical valency known in the chemical
arts.
[0285] Where substituent groups are specified by their conventional chemical
formulae,
written from left to right, they equally encompass the chemically identical
substituents that
would result from writing the structure from right to left, e.g., -CH20- is
equivalent to -OCH2-.
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[0286] The term "alkyl," by itself or as part of another substituent, means,
unless otherwise
stated, a straight (i.e., unbranched) or branched carbon chain (or carbon), or
combination
thereof, which may be fully saturated, mono- or polyunsaturated and can
include mono-, di- and
multivalent radicals, having the number of carbon atoms designated (i.e., Ci-
Cio means one to
ten carbons). Alkyl is an uncyclized chain. Examples of saturated hydrocarbon
radicals include,
but are not limited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-
butyl, t-butyl,
isobutyl, sec-butyl, (cyclohexyl)methyl, homologs and isomers of, for example,
n-pentyl, n-
hexyl, n-heptyl, n-octyl, and the like. An unsaturated alkyl group is one
having one or more
double bonds or triple bonds. Examples of unsaturated alkyl groups include,
but are not limited
to, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl,
3-(1,4-pentadienyl),
ethynyl, 1- and 3-propynyl, 3-butynyl, and the higher homologs and isomers. An
alkoxy is an
alkyl attached to the remainder of the molecule via an oxygen linker (-0-).
[0287] The term "alkylene," by itself or as part of another sub stituent,
means, unless otherwise
stated, a divalent radical derived from an alkyl, as exemplified, but not
limited by, -
CH2CH2CH2CH2-. Typically, an alkyl (or alkylene) group will have from 1 to 24
carbon atoms,
with those groups having 10 or fewer carbon atoms being preferred herein. A
"lower alkyl" or
"lower alkylene" is a shorter chain alkyl or alkylene group, generally having
eight or fewer
carbon atoms. The term "alkenylene," by itself or as part of another
substituent, means, unless
otherwise stated, a divalent radical derived from an alkene.
[0288] The term "heteroalkyl," by itself or in combination with another term,
means, unless
otherwise stated, a stable straight or branched chain, or combinations
thereof, including at least
one carbon atom and at least one heteroatom (e.g., 0, N, P, Si, and S), and
wherein the nitrogen
and sulfur atoms may optionally be oxidized, and the nitrogen heteroatom may
optionally be
quaternized. The heteroatom(s) (e.g., N, S, Si, or P) may be placed at any
interior position of the
heteroalkyl group or at the position at which the alkyl group is attached to
the remainder of the
molecule. Heteroalkyl is an uncyclized chain. Examples include, but are not
limited to: -CH2-
CH2-0-CH3, -CH2-CH2-NH-CH3, -CH2-CH2-N(CH3)-CH3, -CH2-S-CH2-CH3, -CH2-CH2, -
5(0)-
CH3, -CH2-CH2-S(0)2-CH3, -CH=CH-0-CH3, -Si(CH3)3, -CH2-CH=N-OCH3, -CH=CH-
N(CH3)-CH3, -0-CH3, -0-CH2-CH3, and -CN. Up to two or three heteroatoms may be
consecutive, such as, for example, -CH2-NH-OCH3 and -CH2-0-Si(CH3)3. A
heteroalkyl moiety
may include one heteroatom (e.g., 0, N, S, Si, or P). A heteroalkyl moiety may
include two
optionally different heteroatoms (e.g., 0, N, S, Si, or P). A heteroalkyl
moiety may include three
optionally different heteroatoms (e.g., 0, N, S, Si, or P). A heteroalkyl
moiety may include four
optionally different heteroatoms (e.g., 0, N, S, Si, or P). A heteroalkyl
moiety may include five
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optionally different heteroatoms (e.g., 0, N, S, Si, or P). A heteroalkyl
moiety may include up to
8 optionally different heteroatoms (e.g., 0, N, S, Si, or P).
[0289] Similarly, the term "heteroalkylene," by itself or as part of another
substituent, means,
unless otherwise stated, a divalent radical derived from heteroalkyl, as
exemplified, but not
limited by, -CH2-CH2-S-CH2-CH2- and -CH2-S-CH2-CH2-NH-CH2-. For heteroalkylene
groups,
heteroatoms can also occupy either or both of the chain termini (e.g.,
alkyleneoxy,
alkylenedioxy, alkyleneamino, alkylenediamino, and the like). Still further,
for alkylene and
heteroalkylene linking groups, no orientation of the linking group is implied
by the direction in
which the formula of the linking group is written. For example, the formula -
C(0)2R'- represents
both -C(0)2R'- and -R'C(0)2-. As described above, heteroalkyl groups, as used
herein, include
those groups that are attached to the remainder of the molecule through a
heteroatom, such as -
C(0)R', -C(0)NR', -NR'R", -OR', -SR', and/or -502R'. Where "heteroalkyl" is
recited, followed
by recitations of specific heteroalkyl groups, such as -NR'R" or the like, it
will be understood
that the terms heteroalkyl and -NR'R" are not redundant or mutually exclusive.
Rather, the
specific heteroalkyl groups are recited to add clarity. Thus, the term
"heteroalkyl" should not be
interpreted herein as excluding specific heteroalkyl groups, such as -NR'R" or
the like.
[0290] The terms "cycloalkyl" and "heterocycloalkyl," by themselves or in
combination with
other terms, mean, unless otherwise stated, cyclic versions of "alkyl" and
"heteroalkyl,"
respectively. Cycloalkyl and heterocycloalkyl are not aromatic. Additionally,
for
heterocycloalkyl, a heteroatom can occupy the position at which the
heterocycle is attached to
the remainder of the molecule. Examples of cycloalkyl include, but are not
limited to,
cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3 -
cyclohexenyl, cycloheptyl,
and the like. Examples of heterocycloalkyl include, but are not limited to, 1 -
(1,2,5,6-
tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl, 3 -piperidinyl, 4-
morpholinyl, 3-morpholinyl,
tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothien-2-yl,
tetrahydrothien-3-yl, 1-
piperazinyl, 2-piperazinyl, and the like. A "cycloalkylene" and a
"heterocycloalkylene," alone or
as part of another substituent, means a divalent radical derived from a
cycloalkyl and
heterocycloalkyl, respectively. "Cycloalkyl" is also meant to refer to
bicyclic and polycyclic
hydrocarbon rings such as, for example, bicyclo[2.2.1]heptane,
bicyclo[2.2.2]octane, etc.
[0291] The terms "halo" or "halogen," by themselves or as part of another sub
stituent, mean,
unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom.
Additionally, terms such
as "haloalkyl" are meant to include monohaloalkyl and polyhaloalkyl. For
example, the term
"halo(C1-C4)alkyl" includes, but is not limited to, fluoromethyl,
difluoromethyl, trifluoromethyl,
2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like.
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[0292] The term "acyl" means, unless otherwise stated, -C(0)R where R is a
substituted or
unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or
unsubstituted
heteroalkyl, substituted or unsubstituted heterocycloalkyl, substituted or
unsubstituted aryl, or
substituted or unsubstituted heteroaryl.
[0293] The term "aryl" means, unless otherwise stated, a polyunsaturated,
aromatic,
hydrocarbon substituent, which can be a single ring or multiple rings
(preferably from 1 to 3
rings) that are fused together (i.e., a fused ring aryl) or linked covalently.
A fused ring aryl refers
to multiple rings fused together wherein at least one of the fused rings is an
aryl ring. The term
"heteroaryl" refers to aryl groups (or rings) that contain at least one
heteroatom such as N, 0, or
S, wherein the nitrogen and sulfur atoms are optionally oxidized, and the
nitrogen atom(s) are
optionally quaternized. Thus, the term "heteroaryl" includes fused ring
heteroaryl groups (i.e.,
multiple rings fused together wherein at least one of the fused rings is a
heteroaromatic ring). A
5,6-fused ring heteroarylene refers to two rings fused together, wherein one
ring has 5 members
and the other ring has 6 members, and wherein at least one ring is a
heteroaryl ring. Likewise, a
6,6-fused ring heteroarylene refers to two rings fused together, wherein one
ring has 6 members
and the other ring has 6 members, and wherein at least one ring is a
heteroaryl ring. And a 6,5-
fused ring heteroarylene refers to two rings fused together, wherein one ring
has 6 members and
the other ring has 5 members, and wherein at least one ring is a heteroaryl
ring. A heteroaryl
group can be attached to the remainder of the molecule through a carbon or
heteroatom. Non-
limiting examples of aryl and heteroaryl groups include phenyl, naphthyl,
pyrrolyl, pyrazolyl,
pyridazinyl, triazinyl, pyrimidinyl, imidazolyl, pyrazinyl, purinyl, oxazolyl,
isoxazolyl,
thiazolyl, furyl, thienyl, pyridyl, pyrimidyl, benzothiazolyl, benzoxazoyl
benzimidazolyl,
benzofuran, isobenzofuranyl, indolyl, isoindolyl, benzothiophenyl,
isoquinolyl, quinoxalinyl,
quinolyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-
pyrrolyl, 3-pyrazolyl, 2-
imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-
oxazolyl, 5-oxazolyl, 3-
isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl,
2-furyl, 3-furyl, 2-
thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl,
5-benzothiazolyl,
purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl, 2-
quinoxalinyl, 5-
quinoxalinyl, 3-quinolyl, and 6-quinolyl. Substituents for each of the above
noted aryl and
heteroaryl ring systems are selected from the group of acceptable sub
stituents described below.
An "arylene" and a "heteroarylene," alone or as part of another substituent,
mean a divalent
radical derived from an aryl and heteroaryl, respectively. A heteroaryl group
substituent may be
-0- bonded to a ring heteroatom nitrogen.
[0294] Spirocyclic rings are two or more rings wherein adjacent rings are
attached through a
single atom. The individual rings within spirocyclic rings may be identical or
different.
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Individual rings in spirocyclic rings may be substituted or unsubstituted and
may have different
substituents from other individual rings within a set of spirocyclic rings.
Possible substituents
for individual rings within spirocyclic rings are the possible substituents
for the same ring when
not part of spirocyclic rings (e.g. substituents for cycloalkyl or
heterocycloalkyl rings).
Spirocylic rings may be substituted or unsubstituted cycloalkyl, substituted
or unsubstituted
cycloalkylene, substituted or unsubstituted heterocycloalkyl or substituted or
unsubstituted
heterocycloalkylene and individual rings within a spirocyclic ring group may
be any of the
immediately previous list, including having all rings of one type (e.g. all
rings being substituted
heterocycloalkylene wherein each ring may be the same or different substituted
heterocycloalkylene). When referring to a spirocyclic ring system,
heterocyclic spirocyclic rings
means a spirocyclic rings wherein at least one ring is a heterocyclic ring and
wherein each ring
may be a different ring. When referring to a spirocyclic ring system,
substituted spirocyclic rings
means that at least one ring is substituted and each substituent may
optionally be different.
[0295] The symbol "denotes the point of attachment of a chemical moiety
to the
remainder of a molecule or chemical formula.
[0296] The term "oxo," as used herein, means an oxygen that is double bonded
to a carbon
atom.
[0297] The term "alkylarylene" as an arylene moiety covalently bonded to an
alkylene moiety
(also referred to herein as an alkylene linker). In certain embodiments, the
alkylarylene group
has the formula:
6 6
4 4 2 2
3 or 3
An alkylarylene moiety may be substituted (e.g. with a substituent group) on
the alkylene
moiety or the arylene linker (e.g. at carbons 2, 3, 4, or 6) with halogen,
oxo, -N3, -CF3, -CC13, -
CBr3, -CI3, -CN, -CHO, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -S02CH3 -S03H, -
0S03H, -
SO2NH2, ¨NHNH2, ¨ONH2, ¨NHC(0)NHNH2, substituted or unsubstituted C1-05 alkyl
or
substituted or unsubstituted 2 to 5 membered heteroalkyl). In certain
embodiments, the
alkylarylene is unsubstituted.
[0298] Each of the above terms (e.g., "alkyl," "heteroalkyl," "cycloalkyl,"
"heterocycloalkyl,"
"aryl," and "heteroaryl") includes both substituted and unsubstituted forms of
the indicated
radical. Preferred substituents for each type of radical are provided below.
[0299] Substituents for the alkyl and heteroalkyl radicals (including those
groups often
referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl,
cycloalkyl,
heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) can be one or more of
a variety of
groups selected from, but not limited to, -OR', =0, =NR', =N-OR', -NR'R", -
SR', -halogen, -
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SiR'R"R", -0C(0)R', -C(0)R', -CO2R', -CONR'R", -0C(0)NR'R", -NR"C(0)R', -NR-
C(0)NR"R", -NR"C(0)2R', -NR-C(NR'R"R")=NR", -NR-C(NR'R")=NR", -S(0)R', -
S(0)2R', -
S(0)2NR'R", -NRSO2R', -NR'NR"R", -0NR'R", -NR'C(0)NR"NR"R", -CN, -NO2, -
NR'SO2R", -NR'C(0)R", -NR'C(0)-OR", -NR'OR", in a number ranging from zero to
(2m'+1),
where m' is the total number of carbon atoms in such radical. R, R', R", R",
and R" each
preferably independently refer to hydrogen, substituted or unsubstituted
heteroalkyl, substituted
or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,
substituted or
unsubstituted aryl (e.g., aryl substituted with 1-3 halogens), substituted or
unsubstituted
heteroaryl, substituted or unsubstituted alkyl, alkoxy, or thioalkoxy groups,
or arylalkyl groups.
When a compound described herein includes more than one R group, for example,
each of the R
groups is independently selected as are each R', R", R", and R" group when
more than one of
these groups is present. When R' and R" are attached to the same nitrogen
atom, they can be
combined with the nitrogen atom to form a 4-, 5-, 6-, or 7-membered ring. For
example, -NR'R"
includes, but is not limited to, 1-pyrrolidinyl and 4-morpholinyl. From the
above discussion of
substituents, one of skill in the art will understand that the term "alkyl" is
meant to include
groups including carbon atoms bound to groups other than hydrogen groups, such
as haloalkyl
(e.g., -CF3 and -CH2CF3) and acyl (e.g., -C(0)CH3, -C(0)CF3, -C(0)CH2OCH3, and
the like).
[0300] Similar to the substituents described for the alkyl radical,
substituents for the aryl and
heteroaryl groups are varied and are selected from, for example: -OR', -NR'R",
-SR', -halogen, -
SiR'R"R", -0C(0)R', -C(0)R', -CO2R', -CONR'R", -0C(0)NR'R", -NR"C(0)R', -NR-
C(0)NR"R", -NR"C(0)2R', -NR-C(NR'R"R")=NR", -NR-C(NR'R")=NR", -S(0)R', -
S(0)2R', -
S(0)2NR'R", -NRSO2R', -NR'NR"R", -0NR'R", -NR'C(0)NR"NR"R", -CN, -NO2, -R', -
N3, -
CH(Ph)2, fluoro(Ci-C4)alkoxy, and fluoro(Ci-C4)alkyl, -NR' 502R", -NR'C(0)R", -
NR'C(0)-
OR", -NR'OR", in a number ranging from zero to the total number of open
valences on the
aromatic ring system; and where R', R", R", and R" are preferably
independently selected from
hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted
heteroalkyl, substituted
or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,
substituted or
unsubstituted aryl, and substituted or unsubstituted heteroaryl. When a
compound described
herein includes more than one R group, for example, each of the R groups is
independently
selected as are each R', R", R", and R" groups when more than one of these
groups is present.
[0301] Substituents for rings (e.g. cycloalkyl, heterocycloalkyl, aryl,
heteroaryl,
cycloalkylene, heterocycloalkylene, arylene, or heteroarylene) may be depicted
as substituents
on the ring rather than on a specific atom of a ring (commonly referred to as
a floating
substituent). In such a case, the substituent may be attached to any of the
ring atoms (obeying
the rules of chemical valency) and in the case of fused rings or spirocyclic
rings, a substituent
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depicted as associated with one member of the fused rings or spirocyclic rings
(a floating
substituent on a single ring), may be a substituent on any of the fused rings
or spirocyclic rings
(a floating substituent on multiple rings). When a substituent is attached to
a ring, but not a
specific atom (a floating sub stituent), and a subscript for the sub stituent
is an integer greater than
one, the multiple sub stituents may be on the same atom, same ring, different
atoms, different
fused rings, different spirocyclic rings, and each substituent may optionally
be different. Where
a point of attachment of a ring to the remainder of a molecule is not limited
to a single atom (a
floating substituent), the attachment point may be any atom of the ring and in
the case of a fused
ring or spirocyclic ring, any atom of any of the fused rings or spirocyclic
rings while obeying the
rules of chemical valency. Where a ring, fused rings, or spirocyclic rings
contain one or more
ring heteroatoms and the ring, fused rings, or spirocyclic rings are shown
with one more floating
substituents (including, but not limited to, points of attachment to the
remainder of the
molecule), the floating substituents may be bonded to the heteroatoms. Where
the ring
heteroatoms are shown bound to one or more hydrogens (e.g. a ring nitrogen
with two bonds to
ring atoms and a third bond to a hydrogen) in the structure or formula with
the floating
substituent, when the heteroatom is bonded to the floating substituent, the
substituent will be
understood to replace the hydrogen, while obeying the rules of chemical
valency.
[0302] Two or more substituents may optionally be joined to form aryl,
heteroaryl, cycloalkyl,
or heterocycloalkyl groups. Such so-called ring-forming substituents are
typically, though not
necessarily, found attached to a cyclic base structure. In one embodiment, the
ring-forming
substituents are attached to adjacent members of the base structure. For
example, two ring-
forming substituents attached to adjacent members of a cyclic base structure
create a fused ring
structure. In another embodiment, the ring-forming substituents are attached
to a single member
of the base structure. For example, two ring-forming substituents attached to
a single member of
a cyclic base structure create a spirocyclic structure. In yet another
embodiment, the ring-
forming substituents are attached to non-adjacent members of the base
structure.
[0303] Two of the substituents on adjacent atoms of the aryl or heteroaryl
ring may optionally
form a ring of the formula -T-C(0)-(CRR)q-U-, wherein T and U are
independently -NR-, -0-, -
CRR'-, or a single bond, and q is an integer of from 0 to 3. Alternatively,
two of the substituents
on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced
with a substituent of
the formula -A-(CH2),-B-, wherein A and B are independently -CRR'-, -0-, -NR-,
-S-, -5(0) -, -
S(0)2-, -S(0)2NR'-, or a single bond, and r is an integer of from 1 to 4. One
of the single bonds
of the new ring so formed may optionally be replaced with a double bond.
Alternatively, two of
the substituents on adjacent atoms of the aryl or heteroaryl ring may
optionally be replaced with
a substituent of the formula -(CRR'),-X'- (C"R"R")d-, where s and d are
independently integers
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of from 0 to 3, and Xis -0-, -NR'-, -S-, -5(0)-, -S(0)2-, or -S(0)2NR'-. The
substituents R, R',
R", and R" are preferably independently selected from hydrogen, substituted or
unsubstituted
alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted
cycloalkyl,
substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted
aryl, and substituted
or unsubstituted heteroaryl.
[0304] As used herein, the terms "heteroatom" or "ring heteroatom" are meant
to include
oxygen (0), nitrogen (N), sulfur (S), phosphorus (P), and silicon (Si).
[0305] A "substituent group," as used herein, means a group selected from the
following
moieties:
(A) oxo, halogen, -CF3, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -503H, -
504H, -
SO2NH2, -NHNH2, -ONH2, -NHC=(0)NHNH2, -NHC=(0) NH2, -NHSO2H, -NHC= (0)H, -
NHC(0)-0H, -NHOH, -0CF3, -OCHF2, unsubstituted alkyl, unsubstituted
heteroalkyl,
unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl,
unsubstituted
heteroaryl, and
(B) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl,
substituted with at least one
substituent selected from:
(i) oxo, halogen, -CF3, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -S03H, -
SO4H, -
SO2NH2, -NHNH2, -ONH2, -NHC=(0)NHNH2, -NHC=(0) NH2, -NHSO2H, -NHC= (0)H, -
NHC(0)-0H, -NHOH, -0CF3, -OCHF2, unsubstituted alkyl, unsubstituted
heteroalkyl,
unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl,
unsubstituted
heteroaryl, and
(ii) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl,
substituted with at least one
substituent selected from:
(a) oxo, halogen, -CF3, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -503H, -
504H, -
502NH2, -NHNH2, -ONH2, -NHC=(0)NHNH2, -NHC=(0) NH2, -NHSO2H, -NHC= (0)H, -
NHC(0)-0H, -NHOH, -0CF3, -OCHF2, unsubstituted alkyl, unsubstituted
heteroalkyl,
unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl,
unsubstituted
heteroaryl, and
(b) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl,
substituted with at least one
substituent selected from: oxo, halogen, -CF3, -CN, -OH, -NH2, -COOH, -CONH2, -
NO2, -SH, -
503H, -504H, -502NH2, -NHNH2, -ONH2, -NHC=(0)NHNH2, -NHC=(0) NH2, -NHSO2H, -
NHC= (0)H, -NHC(0)-0H, -NHOH, -0CF3, -OCHF2, unsubstituted alkyl,
unsubstituted
heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl,
unsubstituted aryl,
unsubstituted heteroaryl.
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[0306] A "size-limited substituent" or" size-limited substituent group," as
used herein, means
a group selected from all of the substituents described above for a
"substituent group," wherein
each substituted or unsubstituted alkyl is a substituted or unsubstituted Ci-
Czo alkyl, each
substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2
to 20 membered
heteroalkyl, each substituted or unsubstituted cycloalkyl is a substituted or
unsubstituted C3-C8
cycloalkyl, each substituted or unsubstituted heterocycloalkyl is a
substituted or unsubstituted 3
to 8 membered heterocycloalkyl, each substituted or unsubstituted aryl is a
substituted or
unsubstituted C6-Cio aryl, and each substituted or unsubstituted heteroaryl is
a substituted or
unsubstituted 5 to 10 membered heteroaryl.
[0307] A "lower substituent" or" lower substituent group," as used herein,
means a group
selected from all of the substituents described above for a "substituent
group," wherein each
substituted or unsubstituted alkyl is a substituted or unsubstituted C i-C8
alkyl, each substituted
or unsubstituted heteroalkyl is a substituted or unsubstituted 2 to 8 membered
heteroalkyl, each
substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C3-
C7 cycloalkyl, each
substituted or unsubstituted heterocycloalkyl is a substituted or
unsubstituted 3 to 7 membered
heterocycloalkyl, each substituted or unsubstituted aryl is a substituted or
unsubstituted C6-Cio
aryl, and each substituted or unsubstituted heteroaryl is a substituted or
unsubstituted 5 to 9
membered heteroaryl.
[0308] In some certain embodiments, each substituted group described in the
compounds
herein is substituted with at least one substituent group. More specifically,
in some certain
embodiments, each substituted alkyl, substituted heteroalkyl, substituted
cycloalkyl, substituted
heterocycloalkyl, substituted aryl, substituted heteroaryl, substituted
alkylene, substituted
heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene,
substituted arylene,
and/or substituted heteroarylene described in the compounds herein are
substituted with at least
one substituent group. In other certain embodiments, at least one or all of
these groups are
substituted with at least one size-limited substituent group. In other certain
embodiments, at least
one or all of these groups are substituted with at least one lower substituent
group.
[0309] In other embodiments of the compounds herein, each substituted or
unsubstituted alkyl
may be a substituted or unsubstituted Ci-Czo alkyl, each substituted or
unsubstituted heteroalkyl
is a substituted or unsubstituted 2 to 20 membered heteroalkyl, each
substituted or unsubstituted
cycloalkyl is a substituted or unsubstituted C3-C8 cycloalkyl, each
substituted or unsubstituted
heterocycloalkyl is a substituted or unsubstituted 3 to 8 membered
heterocycloalkyl, each
substituted or unsubstituted aryl is a substituted or unsubstituted C6-Cio
aryl, and/or each
substituted or unsubstituted heteroaryl is a substituted or unsubstituted 5 to
10 membered
heteroaryl. In some embodiments of the compounds herein, each substituted or
unsubstituted
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alkylene is a substituted or unsubstituted Ci-Czo alkylene, each substituted
or unsubstituted
heteroalkylene is a substituted or unsubstituted 2 to 20 membered
heteroalkylene, each
substituted or unsubstituted cycloalkylene is a substituted or unsubstituted
C3-C8 cycloalkylene,
each substituted or unsubstituted heterocycloalkylene is a substituted or
unsubstituted 3 to 8
membered heterocycloalkylene, each substituted or unsubstituted arylene is a
substituted or
unsubstituted C6-Cio arylene, and/or each substituted or unsubstituted
heteroarylene is a
substituted or unsubstituted 5 to 10 membered heteroarylene.
[0310] In some certain embodiments, each substituted or unsubstituted alkyl is
a substituted or
unsubstituted Ci-C8 alkyl, each substituted or unsubstituted heteroalkyl is a
substituted or
unsubstituted 2 to 8 membered heteroalkyl, each substituted or unsubstituted
cycloalkyl is a
substituted or unsubstituted C3-C7 cycloalkyl, each substituted or
unsubstituted heterocycloalkyl
is a substituted or unsubstituted 3 to 7 membered heterocycloalkyl, each
substituted or
unsubstituted aryl is a substituted or unsubstituted C6-Cio aryl, and/or each
substituted or
unsubstituted heteroaryl is a substituted or unsubstituted 5 to 9 membered
heteroaryl. In some
certain embodiments, each substituted or unsubstituted alkylene is a
substituted or unsubstituted
Ci-C8 alkylene, each substituted or unsubstituted heteroalkylene is a
substituted or unsubstituted
2 to 8 membered heteroalkylene, each substituted or unsubstituted
cycloalkylene is a substituted
or unsubstituted C3-C7 cycloalkylene, each substituted or unsubstituted
heterocycloalkylene is a
substituted or unsubstituted 3 to 7 membered heterocycloalkylene, each
substituted or
unsubstituted arylene is a substituted or unsubstituted C6-Cio arylene, and/or
each substituted or
unsubstituted heteroarylene is a substituted or unsubstituted 5 to 9 membered
heteroarylene. In
some certain embodiments, the compound is a chemical species set forth in the
Examples
section, figures, or tables below.
[0311] Certain compounds of the present invention possess asymmetric carbon
atoms (optical
or chiral centers) or double bonds; the enantiomers, racemates, diastereomers,
tautomers,
geometric isomers, stereoisometric forms that may be defined, in terms of
absolute
stereochemistry, as (R)-or (S)- or, as (D)- or (L)- for amino acids, and
individual isomers are
encompassed within the scope of the present invention. The compounds of the
present invention
do not include those that are known in art to be too unstable to synthesize
and/or isolate. The
present invention is meant to include compounds in racemic and optically pure
forms. Optically
active (R)- and (S)-, or (D)- and (L)-isomers may be prepared using chiral
synthons or chiral
reagents, or resolved using conventional techniques. When the compounds
described herein
contain olefinic bonds or other centers of geometric asymmetry, and unless
specified otherwise,
it is intended that the compounds include both E and Z geometric isomers.
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[0312] As used herein, the term "isomers" refers to compounds having the same
number and
kind of atoms, and hence the same molecular weight, but differing in respect
to the structural
arrangement or configuration of the atoms.
[0313] The term "tautomer," as used herein, refers to one of two or more
structural isomers
which exist in equilibrium and which are readily converted from one isomeric
form to another.
[0314] It will be apparent to one skilled in the art that certain compounds of
this invention
may exist in tautomeric forms, all such tautomeric forms of the compounds
being within the
scope of the invention.
[0315] Unless otherwise stated, structures depicted herein are also meant to
include all
stereochemical forms of the structure; i.e., the R and S configurations for
each asymmetric
center. Therefore, single stereochemical isomers as well as enantiomeric and
diastereomeric
mixtures of the present compounds are within the scope of the invention.
[0316] Unless otherwise stated, structures depicted herein are also meant to
include
compounds which differ only in the presence of one or more isotopically
enriched atoms. For
example, compounds having the present structures except for the replacement of
a hydrogen by
a deuterium or tritium, or the replacement of a carbon by 13C- or 14C-enriched
carbon are within
the scope of this invention.
[0317] The compounds of the present invention may also contain unnatural
proportions of
atomic isotopes at one or more of the atoms that constitute such compounds.
For example, the
compounds may be radiolabeled with radioactive isotopes, such as for example
tritium (3H),
iodine-125 (1251) or carbon-14 (14C). All isotopic variations of the compounds
of the present
invention, whether radioactive or not, are encompassed within the scope of the
present
invention.
[0318] It should be noted that throughout the application that alternatives
are written in
Markush groups, for example, each amino acid position that contains more than
one possible
amino acid. It is specifically contemplated that each member of the Markush
group should be
considered separately, thereby comprising another embodiment, and the Markush
group is not to
be read as a single unit.
[0319] "Analog" or "analogue" is used in accordance with its plain ordinary
meaning within
Chemistry and Biology and refers to a chemical compound that is structurally
similar to another
compound (i.e., a so-called "reference" compound) but differs in composition,
e.g., in the
replacement of one atom by an atom of a different element, or in the presence
of a particular
functional group, or the replacement of one functional group by another
functional group, or the
absolute stereochemistry of one or more chiral centers of the reference
compound. Accordingly,
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an analog is a compound that is similar or comparable in function and
appearance but not in
structure or origin to a reference compound.
[0320] The terms "a" or "an," as used in herein means one or more. In
addition, the phrase
"substituted with a[n]," as used herein, means the specified group may be
substituted with one or
more of any or all of the named substituents. For example, where a group, such
as an alkyl or
heteroaryl group, is "substituted with an unsubstituted Ci-C20 alkyl, or
unsubstituted 2 to 20
membered heteroalkyl," the group may contain one or more unsubstituted Ci-C20
alkyls, and/or
one or more unsubstituted 2 to 20 membered heteroalkyls.
[0321] Moreover, where a moiety is substituted with an R substituent, the
group may be
referred to as "R-substituted." Where a moiety is R-substituted, the moiety is
substituted with at
least one R substituent and each R substituent is optionally different. Where
a particular R group
is present in the description of a chemical genus (such as Formula (II)), a
Roman alphabetic
symbol may be used to distinguish each appearance of that particular R group.
For example,
where multiple R13 substituents are present, each R13 substituent may be
distinguished as R13A,
Ri3B, Ri3c, Rol), etc., wherein each of R13A, R1313, R13C, R13D, etc. is
defined within the scope of
the definition of R13 and optionally differently.
[0322] Descriptions of compounds of the present invention are limited by
principles of
chemical bonding known to those skilled in the art. Accordingly, where a group
may be
substituted by one or more of a number of sub stituents, such substitutions
are selected so as to
comply with principles of chemical bonding and to give compounds which are not
inherently
unstable and/or would be known to one of ordinary skill in the art as likely
to be unstable under
ambient conditions, such as aqueous, neutral, and several known physiological
conditions. For
example, a heterocycloalkyl or heteroaryl is attached to the remainder of the
molecule via a ring
heteroatom in compliance with principles of chemical bonding known to those
skilled in the art
thereby avoiding inherently unstable compounds.
[0323] The term "pharmaceutically acceptable salts" is meant to include salts
of the active
compounds that are prepared with relatively nontoxic acids or bases, depending
on the particular
substituents found on the compounds described herein. When compounds of the
present
invention contain relatively acidic functionalities, base addition salts can
be obtained by
contacting the neutral form of such compounds with a sufficient amount of the
desired base,
either neat or in a suitable inert solvent. Examples of pharmaceutically
acceptable base addition
salts include sodium, potassium, calcium, ammonium, organic amino, or
magnesium salt, or a
similar salt. When compounds of the present invention contain relatively basic
functionalities,
acid addition salts can be obtained by contacting the neutral form of such
compounds with a
sufficient amount of the desired acid, either neat or in a suitable inert
solvent. Examples of
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pharmaceutically acceptable acid addition salts include those derived from
inorganic acids like
hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric,
monohydrogenphosphoric, dihydrogenphosphoric, sulfuric, monohydrogensulfuric,
hydriodic,
or phosphorous acids and the like, as well as the salts derived from
relatively nontoxic organic
acids like acetic, propionic, isobutyric, maleic, malonic, benzoic, succinic,
suberic, fumaric,
lactic, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic, citric,
tartaric, oxalic,
methanesulfonic, and the like. Also included are salts of amino acids such as
arginate and the
like, and salts of organic acids like glucuronic or galactunoric acids and the
like (see, for
example, Berge et at., "Pharmaceutical Salts", Journal of Pharmaceutical
Science, 1977, 66, 1-
19). Certain specific compounds of the present invention contain both basic
and acidic
functionalities that allow the compounds to be converted into either base or
acid addition salts.
[0324] Thus, the compounds of the present invention may exist as salts, such
as with
pharmaceutically acceptable acids. The present invention includes such salts.
Non-limiting
examples of such salts include hydrochlorides, hydrobromides, phosphates,
sulfates,
methanesulfonates, nitrates, maleates, acetates, citrates, fumarates,
proprionates, tartrates (e.g.,
(+)-tartrates, (-)-tartrates, or mixtures thereof including racemic mixtures),
succinates,
benzoates, and salts with amino acids such as glutamic acid, and quaternary
ammonium salts
(e.g. methyl iodide, ethyl iodide, and the like). These salts may be prepared
by methods known
to those skilled in the art.
[0325] The neutral forms of the compounds are preferably regenerated by
contacting the salt
with a base or acid and isolating the parent compound in the conventional
manner. The parent
form of the compound may differ from the various salt forms in certain
physical properties, such
as solubility in polar solvents. In certain embodiments, compounds of the
present invention
contain both basic and acidic functionalities that allow the compounds to be
converted into
either base or acid addition salts. The neutral forms of the compounds may be
regenerated by
contacting the salt with a base or acid and isolating the parent compound in a
conventional
manner. The parent form of the compounds differs from the various salt forms
in certain
physical properties, such as solubility in polar solvents, but, unless
specifically indicated, the
salts disclosed herein are equivalent to the parent form of the compound for
the purposes of the
present invention.
[0326] In addition to salt forms, the present invention provides compounds,
which are in a
prodrug form. Prodrugs of the compounds described herein are those compounds
that readily
undergo chemical changes under physiological conditions to provide the
compounds of the
present invention. Prodrugs of the compounds described herein may be converted
in vivo after
administration. Additionally, prodrugs can be converted to the compounds of
the present
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invention by chemical or biochemical methods in an ex vivo environment, such
as, for example,
when contacted with a suitable enzyme or chemical reagent.
[0327] Certain compounds of the present invention can exist in unsolvated
forms as well as
solvated forms, including hydrated forms. In general, the solvated forms are
equivalent to
unsolvated forms and are encompassed within the scope of the present
invention. Certain
compounds of the present invention may exist in multiple crystalline or
amorphous forms. In
general, all physical forms are equivalent for the uses contemplated by the
present invention and
are intended to be within the scope of the present invention.
[0328] "Contacting" is used in accordance with its plain ordinary meaning and
refers to the
process of allowing at least two distinct species (e.g. chemical compounds
including
biomolecules or cells) to become sufficiently proximal to react, interact or
physically touch. It
should be appreciated; however, the resulting reaction product can be produced
directly from a
reaction between the added reagents or from an intermediate from one or more
of the added
reagents that can be produced in the reaction mixture.
[0329] The term "contacting" may include allowing two species to react,
interact, or
physically touch, wherein the two species may be a compound as described
herein and a protein
or enzyme. In some embodiments contacting includes allowing a compound
described herein to
interact with a protein or enzyme that is involved in a signaling pathway.
[0330] As defined herein, the term "activation", "activate", "activating" and
the like in
reference to a protein refers to conversion of a protein into a biologically
active derivative from
an initial inactive or deactivated state. The terms reference activation, or
activating, sensitizing,
or up-regulating signal transduction or enzymatic activity or the amount of a
protein decreased
in a disease.
[0331] The terms "agonist," "activator," "upregulator," etc. refer to a
substance capable of
detectably increasing the expression or activity of a given gene or protein.
The agonist can
increase expression or activity 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or
more in
comparison to a control in the absence of the agonist. In certain instances,
expression or activity
is 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold or higher than the
expression or activity in the
absence of the agonist. In certain embodiments, an agonist is a molecule that
interacts with a
target to cause or promote an increase in the activation of the target. In
certain embodiments,
activators are molecules that increase, activate, facilitate, enhance
activation, sensitize, or up-
regulate, e.g., a gene, protein, ligand, receptor, or cell.
[0332] As defined herein, the term "inhibition," "inhibit," "inhibiting," and
the like, in
reference to a protein-inhibitor interaction means negatively affecting (e.g.
decreasing) the
activity or function of the protein relative to the activity or function of
the protein in the absence
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of the inhibitor. In embodiments inhibition means negatively affecting (e.g.
decreasing) the
concentration or levels of the protein relative to the concentration or level
of the protein in the
absence of the inhibitor. In embodiments inhibition refers to reduction of a
disease or symptoms
of disease. In certain embodiments, inhibition refers to a reduction in the
activity of a particular
protein target. Thus, inhibition includes, at least in part, partially or
totally blocking stimulation,
decreasing, preventing, or delaying activation, or inactivating,
desensitizing, or down-regulating
signal transduction or enzymatic activity or the amount of a protein. In
certain embodiments,
inhibition refers to a reduction of activity of a target protein resulting
from a direct interaction
(e.g. an inhibitor binds to the target protein). In certain embodiments,
inhibition refers to a
reduction of activity of a target protein from an indirect interaction (e.g.
an inhibitor binds to a
protein that activates the target protein, thereby preventing target protein
activation).
[0333] The terms "inhibitor," "repressor" or "antagonist" or "downregulator"
interchangeably
refer to a substance capable of detectably decreasing the expression or
activity of a given gene
or protein. The antagonist can decrease expression or activity 10%, 20%, 30%,
40%, 50%, 60%,
70%, 80%, 90% or more in comparison to a control in the absence of the
antagonist. In certain
instances, expression or activity is 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold,
10-fold or lower than
the expression or activity in the absence of the antagonist. An antagonist
prevents, reduces,
inhibits, or neutralizes the activity of an agonist, and an antagonist can
also prevent, inhibit, or
reduce constitutive activity of a target, e.g., a target receptor, even where
there is no identified
agonist. In certain embodiments, inhibitors are molecules that decrease,
block, prevent, delay
activation, inactivate, desensitize, or down-regulate, e.g., a gene, protein,
ligand, receptor, or
cell. An inhibitor may also be defined as a molecule that reduces, blocks, or
inactivates a
constitutive activity. An "antagonist" is a molecule that opposes the
action(s) of an agonist.
[0334] The terms "disease" or "condition" refer to a state of being or health
status of a patient
or subject capable of being treated with the compounds or methods provided
herein. The disease
may be a cancer.
[0335] The terms "treating" or "treatment" refer to any indicia of success in
the therapy or
amelioration of an injury, disease, pathology or condition, including any
objective or subjective
parameter such as abatement; remission; diminishing of symptoms or making the
injury,
pathology or condition more tolerable to the patient; slowing in the rate of
degeneration or
decline; making the final point of degeneration less debilitating; improving a
patient's physical
or mental well-being. The treatment or amelioration of symptoms can be based
on objective or
subjective parameters; including the results of a physical examination,
neuropsychiatric exams,
and/or a psychiatric evaluation. The term "treating" and conjugations thereof,
may include
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prevention of an injury, pathology, condition, or disease. In certain
embodiments, treating is
preventing. In certain embodiments, treating does not include preventing.
[0336] "Treating" or "treatment" as used herein (and as well-understood in the
art) also
broadly includes any approach for obtaining beneficial or desired results in a
subject's condition,
including clinical results. Beneficial or desired clinical results can
include, but are not limited to,
alleviation or amelioration of one or more symptoms or conditions,
diminishment of the extent
of a disease, stabilizing (i.e., not worsening) the state of disease,
prevention of a disease's
transmission or spread, delay or slowing of disease progression, amelioration
or palliation of the
disease state, diminishment of the reoccurrence of disease, and remission,
whether partial or
total and whether detectable or undetectable. In other words, "treatment" as
used herein includes
any cure, amelioration, or prevention of a disease. Treatment may prevent the
disease from
occurring; inhibit the disease's spread; relieve the disease's symptoms, fully
or partially remove
the disease's underlying cause, shorten a disease's duration, or do a
combination of these things.
[0337] "Treating" and "treatment" as used herein include prophylactic
treatment. Treatment
methods include administering to a subject a therapeutically effective amount
of a compound
described herein. The administering step may consist of a single
administration or may include a
series of administrations. The length of the treatment period depends on a
variety of factors,
such as the severity of the condition, the age of the patient, the
concentration of the compound,
the activity of the compositions used in the treatment, or a combination
thereof It will also be
appreciated that the effective dosage of an agent used for the treatment or
prophylaxis may
increase or decrease over the course of a particular treatment or prophylaxis
regime. Changes in
dosage may result and become apparent by standard diagnostic assays known in
the art. In some
instances, chronic administration may be required. For example, the
compositions are
administered to the subject in an amount and for a duration sufficient to
treat the patient.
[0338] The term "prevent" refers to a decrease in the occurrence of disease
symptoms in a
patient. As indicated above, the prevention may be complete (no detectable
symptoms) or
partial, such that fewer symptoms are observed than would likely occur absent
treatment. In
certain embodiments, prevent refers to slowing the progression of the disease,
disorder or
condition or inhibiting progression thereof to a harmful or otherwise
undesired state.
EXAMPLES
[0339] These examples are provided for illustrative purposes only and not to
limit the scope of
the claims provided herein.
Chemical Synthesis Examples
[0340] The compounds in Tables 1-3 are prepared in the following examples.
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TABLE 1
Compound/ UNA
Structure as a part of
UNA Structure
Eg No
oligonucleotide
e,
is 5' End of nucleotide
ODMT
X.--4E' -OH
(:)
0 r_Th....,...,.0
L=,µ.0N NH O,õ."Nõ
1 0 X = 0 or S j [ ' rol
? 1
0."' 0
NC) C)
0 1
3' fid, offmofeotitt,t,1
ODMT NHBznr 0
2,[-. ir
0õ_. rq N
2 0 õ" f7,
0 LO CI): 1 D
O'kN H
NC) 0
,.
ODMT e 6
14.,OcNyNH lky,0
,N.,,ON y NH
0
1õ,..
,k 1 OH
X" OH 1
0 N'
NC) 0 0
1
4
ODMT e Q
r r
14.01NyNH
Li0,õeNyN1-1
4 0 Lo 0
0
,P, 0
0 N' XI -OH LY
NC) HN FEN,
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Compound/ UNA
Structure as a part of
UNA Structure
Eg No oligonucleotide
ODMT e
1%k,,OcNyNH i...., ,0,NH
0 ii
94 1 OL
-,13)õ L
0- N X' / OH =.
NC) li 11
1
ODMT ero c) r---e)
OyNyNH i n
a,- 0
L'O
6
1
1
0-IDNN H 0,
NC)
'9
ODMT 0
1%kOcNyNH LIõO N , ,ISIti
7 0 i If
9 1 F
,P, 0
0 N --P=
NC) X' OH
.t.
ODMT er
Q I
h:NyNH
8 (1 0
T F Cr N :,!'f''0
v
0,P,N 1
--",.
NC) X' OH
ODMT r NHAc cL.---
N, -iA
'1*4*
ON N
9 - Lo
00
,
0-kN H
X' OH
NC) 0
0 N,
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Compound/ UNA Structure as a part of
UNA Structure
Eg No
oligonucleotide
ODMT
NiBu
nr 0 El- if
ON N
1.,,,,õ.e..10, NõN
9
0 --r IT
0
0.} õ 0
0
i
H -:.-P
NC 0 X 'OH
L\I
NDMT
0-- -NH -.;`\=
r 'r
14.,OcNI.r NH
11 9 1 9 0
NC 0 X - '$ OH
1
,--N ----N
//Z-NHBz if
N j-
N $
6 .------. ,
0 DMT
OcN...."
12 3 L
,.... 0
ox i .0,- ....õ, ......,
9 1 oH
P, .....k
NC) C)
--N
i N ,
ODMT o
13 ON_____N
CV"' Ll `s 9 1 soH
,,& I
NC C)
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Compound/ UNA
Structure as a part of
UNA Structure
Eg No oligonucleotide
( NMAIT r'N'sr HIBU 0
1. r L0 II I
i . N
14 0 i ., L0 8
-µ L&
0t.-0
x
,.
NC J .I, 0 0
.....,õ , , -,. -,
TABLE 2
Compound/ UNA
Structure as a part of
Structure
Eg No oligonucleotide
ODMT
1440eNl.rNH
150 0 (R1A)n
NC) X 'OH 1
-,=,----I
TABLE 3
Compound/ UNA
Structure as a part of
Structure
Eg No oligonucleotide
RiB0 ......0
OeNl.rNH
il
16 L 0 0) ,0 0
9 1 OH
,13
0 N ,
X
t
NC)5' Ea/41 ewboty '
Example 1. General synthetic scheme for the preparation of Compounds 1-13, UNA
2' derivatives:
[0341] Compounds 1-15 are synthesized according to the following schemes
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ODMT ODMT ODMT
0..=Base Bzcl, CH2Cl2
LOH 50(Base 1. TBSCI, Pyridine
______________________________________________________ 3.--
2. NaOH,Me0H LO.,,õBase
LOH
0
HO HO TBSO
0 0
ODMT ODMT
LO..õBase Lx0d.Base
R-X, NaH, DMF
L OR 1. NH4F OR
______________________________________________ ).-
TBSO 2.
P,
N C:r N
O'kNJ\ NC)
NC)
Base =
e\r0 NHBz N)4 ii¨N
¨NHBz
ts(NyNH Ns(NyN c0
HN
0 ) 0 µ,.(1\1NN /¨NH
Me
N0
\r0
Ns(NirNH ts(NNN
0
R-X = 0 0
H
Br Br Br , Br
B 0 Br Br
Br
Example 2. General Synthetic Scheme for UNA 2' Fluoro Compounds:
[0342] Compounds 2 and 8 are synthesized according to the following schemes.
ODMT ODMT
ODMT
C:ocOH F Base Triflic anhydride
TBSO , Py
(:) Base 1. NH4F 0Base
L)
L
___________________________________________________________ )- Lx
CS2F, DMF TBSO 2. I j_ 0 1 F
P,
P,
:rii 1
0 N- CY N
NC)
NC)
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ODMT ODMT ODMT
5O Base O.
Base e \
Oxidation -.õ... BF4 00,N=SF2 0:(Base
_,_
TBSO LOH ,.,
TBSO H-0 TBSO F F
i
O.R.N
ODMT
ODMT NC) rOxBase LOroBase
L)....-
HO F F OF )F)F
I
0" N
NC)
Example 3. General Synthetic Scheme for the Solid Phase Synthesis of
Oligonucleotides:
,DMT
DMT 0 DMT
\....0' ....... Base 0'
\ft...O....Base / \ft...O....Base
0 -IR
do IR
HO 1 N9i IR I
1CN 1
X=P, CN
,P(:)
,CN
\b....O....Base 0 w I 0
0
d .1,
________________________________________________________________ 1
___________________ b.- = -,
, Oxidation Deprotection
6 Coupling d R
o ..., .:.
d R
ox = S or 0
DMT DMT
0' o'
\ft.......Base \ft.......Base
0 R
i 9 R
HO
1.....(:)...Base DMT 1 0
0 I 0
0 DMT
0' 0'
\ft...O....Base \ft...O....Base
$ '-, µ.........Base
\..,......Base
d R
X=P0 , .CN 0 R d -R
I ? R I I
0 1
CN X=P0, CN
I X =1:13,0....--,....õ,.CN
? R
L.O.Base . P
'N- 0 0 0 N,P,0CN
\...õ(19).0Base \ft...O....Base
do IR
o Coupling %
0 R
6 Oxidation
õ
,
0 R 1. Coupling
2. Oxidation
o
X = S or 0
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DMT
o'
lõ,..c) z. Base HO
\.,..Or Base
0 R
1 0 P, CN R
I 0 I Phosphodiester
0 0=P¨OH -4-
1 internucleotide
z=Base 0 linkage
\....,,,=Base
0 R
0=1P Cleavage and 0 R
, 1
1 OH deprtection 0=19,
0 to get oligonucleotide I OH
LO=Base _________________________ = 0
\,....O0 Base
'-
0
1 0 IR
X=P,
I OH 1
0 X=P,
I LO OH0Base 0
LO0Base
$ '-
X = S or 0 d -R $ =.
o
HO . -
R
/
Oligonucleotide could be further lengthened by
repeating additional cycles of coupling and oxidation
Example 4. UNA key
TABLE 4
Structure Modification Name Code
UNA with 2' -MOE Uq
D.Nrroey). t'4 yN-
\to
UNA with 2' -EOE Uw
Dieite:r : i
0
hr,,,,...."..k,v0,4õ,4 = .
1
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n UNA with 2'-methyl Ux
tr ti
OMT rey0 .(t) . . . acetamide
k
0 -
4, Lro ..."......,..:.po. UNA
with 2'-mPEG2 Uy
r 1
a
t4 .'',V6e4kNrA.4)sb'k= . ..
. .
oteANc 04,,,t.
1
Example 5. Synthesis of Uridine-UNA-2'MOE, 3'Phosphoramidite
[0343] Uridine-UNA-2'MOE, 3'Phosphoramidite is synthesized according to the
following
schemes.
Na104 -
--"'=-,-;=-
DWETrCI r K
_L...., /-'_,_. - i djoxane, O. 25
C, 2 h Dtare*T N'it-- ¨.1'1 Py,0-25 C, 3 h
Hd 'OH 0 Fr' -OH 0
-, ''0
6
crude
79.2% yield
1 2 3
r--- - = . . . ,r Lipmerke TLIM MO% WI (Y-
NaBH4
111%.11
LWITIO' 44'1' .µk=Croly 'NH ............................. yõ,NIõ NH o-
&vane, 0- 20 'C, 1 h 0 benzoyl ben2oate, id. Py 25 C.
8 h
Ha=-= l-i 20-40 *C. 4 ti HO-- L'OBz
09.3% yield aude 44.5%
yieici
4 5
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f
:....,.,,,,,,..o
0
r'r
.,.T
if-
!, 8 N1-14F
: ii (60% purity) MO` -0 Me0H, 20-40
C. 16 h 1-10-
TBSOF COH DMF,0-10 C, 16 h
LI 59.4% yield
36.1% *lc 0
=-, '..
6 7 11
: 4
.--1--, b 0/1Tr0---)="r` g
v.-
DO; DeN1,3-25 C, 3 h
N0.õ...õ.--,0õ
32 6% yield .
..---1,-õ 0-..
U.UNA.TMOE 3'Phosphoramidite
Example 6. Synthesis of Cytosine-UNA-2'MOE, 3'Phosphoramidite
[0344] Cytosine-UNA-2'MOE, 3'Phosphoramidite is synthesized according to the
following
schemes.
nr NHBz
Et3N, DMAP, TPSCI
yNH N yN DMTr0"..4)--
CkYµNyN
DMTr0---.4"--' 'Y'µN NH3 H20 (30%) DMTr0 BzCI
L0 ].. __________ iz:1 0 o 0 __________________ l'""- "---
TBS0*--- TBSO c:1 TBSO
crude 74.3% yield H
DCM, 25 C, 3 h
Py, 0-25 C, 2 h
H
0 0 0
7 8 9
)-
NHBz
......k1 III NHBz
rr nr
.N,R0.-.......CN
TBAF
DMTre.43NyN b DMTre..."-"-a.yµNyN
________ A.-- 0
0 HO _______________________________________ ).- CK 1 0
THF, 0-25 C, 16 h
H DCI, DCM, 0-25 C, 1 h 1
NC,....õ....-,0,,P,N,..).õ. H
yield: 84,8% 36.6% yield
0
0
C-UNA-2'MOE 3'Phosphoramidite
Example 7. Synthesis of Adenine-UNA-2'MOE, 3'Phosphoramidite
[0345] Adenine -UNA-2'MOE, 3'Phosphoramidite is synthesized according to the
following
schemes.
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or-N, -N 1
=ks.---)11-1R?.!
N' )-.-Nil[k. N ."---NHBz N, i
DMI$C1 ;fil Na104
ea,õõolki N ,A,,,,CL
2 h DMIra At. F14.'"4 Cloxartm1120,
25 T, '1 h ...-4,.. 0 .,=47
MIRO- '''=-=""' . y'r
= - ,
, , crude rtifile
H d OH lid .til 0
12 13 14
= N -N
di- rt4HB2.'= Nii- \ -"
NaB1-1,4 \-er,- PA-CI, DE113 'MS((
inidazola
______________ s-=
l 0 r .
C. ______________________________________________________________________ si.
Clioxatio4420( 6..12) , 26 . 1 h Dmi-ox=-***1- -
'"=-=.=.enN DM, JD '0, / h h
EiMTilYANT." X
46A% yield ctodo crude
for 3 steps h10---- H 110- Otlz
15 16
if 'S. 441.-illx
Ikf. r
-N
.1; "_-NI-lEiz- I) ..;,,r-N1-1Bz
.r \ Na0H(1k0 N.4---1=:"(..
DM-frO".%"(EIL"C-'4V Pylvie0H(2:1)õ DMTrO '-'4.)"-Ct
TN.'" ME., 0-15 "C, 16 h 1660")
"0
crude
r6Se c.)Bz 21.7% yield rBscy N-oli
1
for 3 stgis 0..õ
17 18 19
--". -14¨ r-N
õ..-N ; i ti %---Nl-l'az
it =:''. -NHI3z
N I .... ).., N , .P . , 0 ,,-. .. , ....c.N
Ns_ i
n 0 }.,,N ..).-..,
NH4F, TEA
.
rii4Tr0"-44"Y"-- ."-.....'' f.)01 D,l=Kir0-'4'y ---e.' ---4..
hie0H. 15 C,. 16 h ) DOM, 0-15 C, 2 h
0.-- 1 ...õ0
28.6% yield 1, 34.2% yield
tor 2 steps
1 1
--,
20 A-IAA-MOE 3Thosp1 own i dite
Example 8. Synthesis of Guanosine-UNA-2'MOE, 3 'Phosphoramidite
[0346] Guanosine -UNA-2'MOE, 3'Phosphoramidite is synthesized according to the
following schemes.
/
, ----( õ.
\?P--N1-1 ,,=,--1s11-4
0 V ---\,-11 H
si-N% d s----N .4 S Z 0
1,i 1-1 ;--- NH r , .\_0
N N
Na104 \-----'''N
p 0_15 C. 2 1., DIATt-0' ' 1- . Nr...01
\-:-'N Dioxarre.,H20, 15 C. 1 h
Ha' ;oil L.
crude crude 0' '0
fic.f bli
21 22 23
6 F .0 0
NE,E3N4 ,,, N , DB13, B dzCI N. .
TBSCI, imidazole,
).=
Dioxane, 16 "C, 1 h 1 Ai DCM. -78 C., 1 f-
; DCM, 15 '''C, 2 il
60.3% yield Dly1T10-.4)---C-"eN-/ crude DMIrO''... ny- ----C-
L"'. crude
kii. 3 steps ..---.
HO OH HO-/ Ofilz.
24 26
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_ i vi
¨ ,
, \r-N H
/ H
i ,- N. .
--)r-N, ,-.111 ),---N H a 0 ti µF-----0
/I -N rtr,,,,,n,-
d `,,7 -,ro 0 Nn .1)... 1 14
N Na01-1 (1 M) NuF1
\f---- __________________ Iv-
'r \
kieORPy
, ,i1 ;PI 0 . _...,;,..;,NI OMF,
0 't, lb min .-- -...
1)&41'10 ''''-=.`"DkiTrO'.1'. 'IAN
TBSO 0
Grude
21.6% yield
TBS0" ``Oilz TBSe ''OE-1
for 3 steps
0õ
26 27 28
4 I-I
õ-=N H ). ..1, b ---I: H
II-
0 ,7 \F-..,0
,i, ., Põ-, ,oN )r-N -214
N. ,, -.N- 0 - 6 # \,-...o
,----=:\ N f
NH4, TEA i N ,,,L., OCI -:---
'(
fs.1e0H, 15-4D DCM, 0-15 C, 211
,i l=
38.0% yield HO '9 31.4% yield )
, ..
far 2 skvs 0 0
-..
1 NC õ....,..,0., 4. N.,:c 1...,1
0õ
6.
.,..j.,,,, õ
29
G-UNA-MOE 3'Pliosphotamidtte
Example 9. General procedure for preparation of compound 2
0 0
H0/461%.\7 t DMTrCI DMTr0/464%.( N\r )r_NH
Py,0-25 C, 3 h
HO' -bH HO' bH
79.2% yield
1 2
[0347] To a solution of compound 1(350 g, 1.43 mol, 1.00 eq) in Py (2.1 L),
was added
DMTC1 (500.2 g, 1.48 mol, 1.03 eq) at 0 C. The reaction mixture was stirred
at 25 C for 3 h.
TLC (Dichloromethane: Methanol = 10: 1, Rf = 0.51) showed no starting
material. The mixture
was diluted with aq.NaHCO3 (2.0 L) and extracted with DCM (2.0 L x 2), dried
over anhydrous
Na2SO4, filtered and concentrated under reduced pressure to give a residue.
Concentrate the
combined organic layer to give the crude product. The residue was purified by
column
chromatography (SiO2, Petroleum ether/Ethyl acetate = 10/1 to 0/1 and 0.5%
TEA). Afforded
compound 2 (620 g, 1.13 mol, 79.2 yield) as yellow solid.
Example 10. General procedure for preparation of compound 3
r------\ro
0 Na104
DMTr0Ctirq\¨NH
// dioxane, H20, 25 C, 2 hil. DMTrO N y NH
Hd b H j L 0
0 0
crude
2 3
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[0348] To a solution of compound 2 (310 g, 567.2 mmol, 1.00 eq) in dioxane
(1.5 L), was
added NaI04 (121.3 g, 567.2 mmol, 31.4 mL, 1.00 eq) and H20 (600 mL) at 25 C,
the reaction
mixture was stirred at 25 C for 2 h. TLC (Dichloromethane: Methanol = 10: 1,
Rf = 0.64)
showed starting material was consumed completely. The two reactions were
combined for
workup. The reaction mixture was filtered. The crude product was used into the
next step
without further purification. Afforded compound 3 (600 g, crude) as a
colorless oil.
Example 11. General procedure for preparation of compound 4
NaBH4
DMTrO' 'Y yNH __________________________ DMTre4440eNNH
dioxane, 0- 20 C, 1 h y
0
HO L01-18
99.3% yield
3 4
[0349] To a solution of compound 3 (300 g, 550.9 mmol, 1 eq) in dioxane (1.8
L), was added
NaBH4 (20.8 g, 550.9 mmol, 1.00 eq) at 0 C, the mixture was stirred at 20 C
for 1 h. TLC
(Dichloromethane: Methanol = 10: 1, Rf = 0.43) showed no starting material.
The two reactions
were combined for workup; the reaction mixture was quenched by acetone,
neutralized with
20% acetic acid aq., and concentrated to give a residue. The residue was
diluted with DCM (1.8
L), and washed with H20 (1.8 L), dried over anhydrous Na2SO4, filtered and
concentrated under
reduced pressure. Afforded compound 4 (600 g, 1.09 mol, 99.3% yield) as white
solid.
Example 12. General procedure for preparation of compound 5
rro
r
Lipozyme TLIM (60% W)
DMTrOA%OlgyNH ___________________________________ DMTrO
OHO benzoyl benzoate, tol.
HO 0
HO
20-40 C, 4 h OBz
crude
4 5
[0350] To a solution of compound 4 (300 g, 546.9 mmol, 1.00 eq) in Tol. (600
mL) was added
lipozyme TL IM (60 g, 0.60 eq) and Benzoyl benzoate (124.9 g, 552.3 mmol,
104.1 mL, 1.01
eq) at 20 C. The reaction mixture was stirred at 40 C for 4 h. TLC
(Dichloromethane:
Methanol = 10: 1, Rf = 0.53) showed no starting material. Combined the two
reactions for
workup, the reaction mixture was filtered, the filter liquor was partitioned
between aqueous
NaHCO3 (600 mL) and Et0Ac (600 mL). The organic phase was separated, washed
with brine
(600 mL x 3), dried over anhydrous Na2SO4, filtered and concentrated under
reduced pressure.
Afforded compound 5 (620 g, crude) as white solid.
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Example 13. General procedure for preparation of compound 6
y NH TBSCI
DMTrO0NyNH
DMTrO7C)N
HO 0 Py ,25 C, 8 h
TBSO OHO
OBz
44.5% yield
6
[0351] To a solution of compound 5(310 g, 475.0 mmol, 1.00 eq) in Py (1.8 L)
was added
TBSC1 (85.9 g, 569.9 mmol, 69.8 mL, 1.20 eq). The reaction mixture was stirred
at 25 C for 8
h. Two batches were combined for working up and purification. Aq.NaHCO3 (1.8
L) was
added and the mixture was extracted with DCM (1.8 L), concentrated in vacuum
to give a
residue. The residue was dissolved in Py (1.2 L) and Me0H (5.4 L), then added
1M NaOH in
Me0H (300 mL) dropwise in mixture at 0 C, and then stirred for 1 h at 0 C.
TLC (Petroleum
ether: Ethyl acetate = 1:1, Rf = 0.43) showed no starting material. Sat. aq
NH4C1 (1.2 L) was
added to the mixture, which was stirred for 10 min, H20 (1.2 L) was added and
the mixture
which was extracted with DCM (3 L). Dried over anhydrous Na2SO4, filtered and
concentrated
under reduced pressure to give a residue. The residue was purified by column
chromatography
(5i02, Petroleum ether/Ethyl acetate=10/1 to 0/1 and 0.5% TEA). Afforded
compound 6 (280 g,
422.4 mmol, 44.5% yield) as yellow oil.
Example 14. General procedure for preparation of compound 7
r.r0 a
y NH
DMTre444"N
yNH __________________________________________________ 0
DMTrO N
NaH (60% purity) TBSO 0
O
TBSO OH
DMF,0-10 C, 16 h
36.1% yield
6 7
[0352] To a solution of NaH (27.9 g, 697.0 mmol, 60% purity, 3.30 eq) in DIVIF
(850 mL),
was added compound 6 (140 g, 211.2 mmol, 1.00 eq) at 0 C, the reaction
mixture was stirred at
0 C for 1 h, and then was added compound a (44.0 g, 316.8 mmol, 29.8 mL, 1.50
eq) at 0 C,
the reaction mixture was stirred at 10 C for 16 h. TLC (Petroleum ether:
Ethyl acetate = 1: 2,
Rf = 0.43) showed no starting material. Two batches were combined for working
up and
purification. Dilute the residue with aq.NH4C1 (4.2 L), and extracted with
Et0Ac (1.0 L x 2),
combined organic layers were washed with brine (1.0 L), dried over anhydrous
Na2SO4 and
filtered. The filtrate was concentrated under reduced pressure to give a
residue. The residue
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were purified by column chromatography (SiO2, Petroleum ether/Ethyl acetate =
10/1to 0:1 and
0.5% TEA).Afforded compound 7(110 g, 152.6 mmol, 36.1% yield) as yellow oil.
[0353] 11-1 NMR: ET32436-23-P1A (400 MHz, DMSO-d6)
[0354] 6 11.37 (s, 1H), 7.70 (d, J= 8.0 Hz, 1H), 7.33-7.30 (m, 4H), 7.19-7.16
(m, 5H), 6.86-
6.84 (m, 4H), 6.00 (t, J= 4.8 Hz, 1H), 5.55 (d, J= 8.0 Hz, 1H), 3.73 (s, 6H),
3.71-3.69 (m, 2H),
3.65-3.63 (m, 2H), 3.56-3.55 (m, 3H), 3.41-3.39 (m, 2H), 3.21 (s, 3H), 2.97-
2.95 (m, 2H), -0.76
(s, 9H), -0.04 (d, J= 7.6 Hz, 6.0H)
Example 15. General procedure for preparation of compound 11
r-ro rr0
DIVITre%4N y NH DMTre444 N y""
NH4F
L.0
TBSO HO 0
Me0H, 20-40 C, 16 h
59.4% yield
0 0
7 11
[0355] To a solution of compound 7 (15 g, 20.8 mmol, 1.00 eq) in Me0H (120 mL)
was
added NH4F (6.17 g, 166.5 mmol, 8.00 eq) at 20 C, the reaction mixture was
stirred at 40 C
for 16 h. TLC (Petroleum ether: Ethyl acetate = 0:1, Rf = 0.13) showed no
starting material.
The reaction mixture was concentrated under reduced pressure to give a
residue. The residue
was purified by column chromatography (SiO2, Petroleum ether/Ethyl acetate =
10/1to 0:1 and
0.5% TEA). Afforded compound 11 (7.50 g, 12.4 mmol, 59.4% yield) as yellow
oil.
[0356] 11-1 NMR: ET32436-28-P1A (400 MHz, DMSO-d6)
[0357] 6 11.39 (s, 1H), 7.68 (d, J= 8.0 Hz, 1H), 7.32-7.28 (m, 4H), 7.20-7.16
(m, 5H), 6.87-
6.85 (m, 4H), 5.98 (t, J= 6.0 Hz, 1H), 5.54 (d, J= 4.0 Hz, 1H), 4.74 (t, J=
5.2 Hz, 1H), 3.73 (s,
7H), 3.71 (s, 1H), 3.63-3.59 (m, 1), 3.51-3.55 (m, 2), 3.42-3.41 (m, 4H), 3.21
(s, 3H), 3.00-2.94
(m, 2H).
Example 16. General procedure for preparation of U-UNA-2'MOE 3'Phosphoramidite
)-N-L ro
rr r
o
DMTre6NIINEI b DMTrO N y
LO
HO HDCI, DCM,0-25 C, 3 h
32.6% yield 0 0
11 U-UNA-2'MOE 3'Phosphoramidite
[0358] To a solution of compound 11(8.0 g, 13.2 mmol, 1.00 eq) in DCM (48.0
mL) was
added 1H- imidazole-4,5-dicarbonitrile (2.34 g, 19.8 mmol, 1.50 eq) at 0 C,
and then compound
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b (6.76 g, 22.4 mmol, 7.12 mL, 1.70 eq) was added. The reaction mixture was
stirred at 25 C
for 3 h. TLC (Dichloromethane: Me0H = 10:1, Rf= 0.53) showed no starting
material. The
reaction mixture was purified by column chromatography (SiO2, Petroleum
ether/Ethyl
acetate = 2/1-1/2, 1% TEA). Combine with (ET32436-32-P1) for purification:
Column: Agela
DuraShell C18 250*80mm*10um; mobile phase: [water (10mM NH4HCO3)-ACN]; B%: 40%-
80%, 20min. Afforded compound U-UNA-2'MOE 3'Phosphoramidite as yellow solid.
[0359] 111 NMR: (400 MHz, DMSO-d6): 6 11.40 (br s, 1H), 7.68 (d, J= 8.0 Hz,
1H), 7.32-
7.29 (m, 4H), 7.20-7.16 (m, 5H), 6.86-6.84 (m, 4H), 5.99-5.96 (m, 1H), 5.56
(d, J= 8.0 Hz, 1H),
3.73 (s, 7H), 3.70-3.65 (m, 4H), 3.56-3.55 (m, 5H), 3.40-3.39 (m, 2H), 3.21
(s, 3H), 3.09-2.94
(m, 2H), 2.72-2.69 (m, 2H), 1.17-0.96 (m, 12H).
[0360] 31PNMR: (162 MHz, DMSO-d6): 6 147.46
[0361] HPLC: 97.3% purity
[0362] MS: M-1 = 805.3
Example 17. General procedure for preparation of compound 8
HN 2
I
()
Et3N, DMAP, TPSCI
DMTrO(DyNyN
DMTrO NH3.H20 (30%)
0
L 0 TBSO 0
TBSO 0 DCM, 25 C, 3 h
crude
0
0
7 8
[0363] To a solution of compound 7 (50 g, 69.4 mmol, 1.00 eq) in DCM (300 mL)
and TEA
(17.6 g, 173.4 mmol, 24.13 mL, 2.50 eq), was added DMAP (8.47 g, 69.4 mmol,
1.00 eq) and
TPSC1 (51.1 g, 173.4 mmol, 2.50 eq). The reaction mixture was stirred at 25 C
for 1 h, and
then was added NH3.H20 (81.03 g, 693.6 mmol, 89.0 mL, 30% purity, 10.0 eq) and
stirred at 25
C for 2 h. TLC (Dichloromethane: Methanol = 10: 1, Rf = 0.23) showed no
starting material.
The reaction mixture was extracted in Et0Ac (300 mL) and washed with
sat.NaHCO3 and brine
(200 mL each). Organic layer was dried over anhydrous Na2SO4, filtered and
concentrated
under reduced pressure to give a residue. Afforded compound 8 (50 g, crude) as
yellow oil.
Example 17. General procedure for preparation of compound 9
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FIN 2 NHBz
DMTr0 . N yN BzCI DMTrO 4=1'yN
TBSO Lc) 0
TBSO Lo 0
Py, 0-25 DC, 2 h
74.3% yield
0 0
8
9
[0364] To a solution of compound 8 (40 g, 55.6 mmol, 1.00 eq) in Py (240 mL),
was added
BzCl (11.7 g, 83.3 mmol, 9.68 mL, 1.50 eq) at 0 C, the reaction mixture was
stirred 25 C for 2
h. TLC (Dichloromethane: Methanol = 10: 1, Rf = 0.43) showed no starting
material. The
mixture was diluted with aq.NaHCO3 (500 mL) and extracted with DCM (500 mL x
2), dried
over anhydrous Na2SO4, filtered and concentrated under reduced pressure to
give a residue.
Concentrate the combined organic layer to give the crude product. The residue
was purified by
column chromatography (SiO2, Petroleum ether/Ethyl acetate = 10/1 to 0/1 and
0.5% TEA).
Afforded compound 9 (34.0 g, 41.3 mmol, 74.3% yield) as yellow oil
[0365] 111 NMR: (400 MHz, DMSO-d6) 6 11.36 (s, 1H), 8.58-8.57 (m, 1H), 8.20-
8.18 (m,
1H), 8.04-8.02 (m, 2H), 7.64-7.51 (m, 3H), 7.38-7.15 (m, 12H), 6.86-6.83 (m,
4H), 6.16 (t, J=
5.2 Hz, 1H), 3.73-3.69 (m, 4H), 3.67 (s, 6H), 3.60-3.52 (m, 4H), 3.42-3.39 (m,
2H), 3.21-3.19
(m, 3H), 3.00-2.99 (m, 2H), 0.77 (s, 9H), -0.03 (d, J= 8.0Hz, 6H).
Example 18. General procedure for preparation of compound 10
NHBz NHBz
DMTrO Th' N yrs' DrsATro"-Ny"
TBAF
0 Dm-
0
HO 0 TBSO 0
THF, 0-25 C, 16 h
yield: 84.8%
0 0
9 10
[0366] To a solution of compound 9 (30 g, 36.4 mmol, 1.00 eq) in THF (300 mL),
was added
TBAF (1 M, 40.1 mL, 1.10 eq) at 0 C, the reaction mixture was stirred at 25
C for 16 h. TLC
(Dichloromethane: Methanol = 10: 1, Rf = 0.24) showed no starting material.
The mixture was
diluted with aq.NaHCO3 (600 mL) and extracted with DCM (500 mL x 2), dried
over anhydrous
Na2SO4, filtered and concentrated under reduced pressure to give a residue.
Concentrate the
combined organic layer to give the crude product. The residue was purified by
column
chromatography (SiO2, Petroleum ether/Ethyl acetate = 10/1 to 0:1 and 0.5%
TEA). Afforded
compound 10 (16.5 g, 23.3 mmol, 63.9% yield) as yellow oil.
[0367] 111 NMR: (400 MHz, DMSO-d6) 6 11.36 (s, 1H), 8.20 (d, J= 7.2 Hz, 1H),
8.03 (d, J=
7.6 Hz, 2H), 7.64 (t, J= 7.4 Hz, 1H), 7.55-7.51 (m, 2H), 7.37 (d, J= 7.2 Hz,
1H), 7.31-7.29 (m,
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4H), 7.17 (d, J=8.8 Hz, 5H), 6.86 (d, J= 8.8 Hz, 4H), 6.17 (t, J= 4.8Hz, 1H),
4.77 (t, J= 5.4Hz,
1H), 3.76-3.74 (m, 2H), 3.68 (s, 7H), 3.60-3.58 (m, 2H), 3.46-3.39 (m, 4H),
3.20 (s, 3H), 3.05-
2.96 (m, 2H).
Example 19. General procedure for preparation of C-UNA-2'MOE 3'Phosphoramidite
NHBz )'N'L NHBz
DMTrO(DyNy" DNATroyNy"
He 0 0
DCI, DCM, 0-25 C, 1 h
36.6% yield NC 0,11',N H
0 0
C-UNA-2'MOE 3'Phosphoramidite
[0368] To a solution of compound 10 (13.3 g, 18.7 mmol, 1.00 eq) in DCM (130
mL) was
added 1H-imidazole-4,5-dicarbonitrile (3.32 g, 28.1 mmol, 1.50 eq) at 0 C,
and then compound
b (9.60 g, 31.9 mmol, 10.1 mL, 1.70 eq) was added. The reaction mixture was
stirred at 25 C
for 1 h. TLC (Petroleum ether: Ethyl acetate = 1:1, Rf = 0.43) showed no
starting material. The
reaction mixture was purified by column chromatography (SiO2, Petroleum
ether/Ethyl
acetate = 1/1-0/1, 5% TEA). Afforded compound C-UNA-2'MOE 3'Phosphoramidite as
white solid.
[0369] 111 NMR: (400 MHz, DMSO-d6)
[0370] 6 11.37 (s, 1H), 8.19 (d, J= 6.8 Hz, 1H), 8.03 (d, J= 7.6 Hz, 2H), 7.64
(t, J= 7.4 Hz,
1H), 7.53 (t, J= 7.6 Hz, 2H), 7.36 (d, J= 7.2 Hz, 1H), 7.29-7.26 (m, 4H), 7.18-
7.15 (m, 5H),
6.85 (d, J= 8.4 Hz, 4H), 6.15 (s, 1H), 3.87-3.68 (m, 11H), 3.64-3.57 (m, 4H),
3.54-3.44 (m,
2H), 3.40 (s, 2H), 3.20 (s, 3H), 3.12-2.30 (m, 2H), 2.74-2.69 (m, 2H), 1.12-
1.10 (m, 6H), 1.05-
1.01 (m, 6H)
[0371] 31PNMR: (162 MHz, DMSO-d6): 6 147.57
[0372] HPLC: 98.4% purity
[0373] MS: M-1 = 909.3
Example 20. General procedure for preparation of compound 13
N N
DMTrCI )-
0 0
N N
H0/46.6.. Py, 25 C, 2 h DMTr0/46*.1 N N
He. .-0H HOs -10H
12 13
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[0374] To a solution of compound 12 (200 g, 539 mmol, 1.00 eq) in Py (2.00 L)
was added
DMTC1 (200 g, 592 mmol, 1.10 eq), then stirred at 25 C for 2 h. TLC
(Dichloromethane:
Methanol = 20: 1, Rf= 0.44) showed no starting material. The reaction mixture
was added
Me0H (150 mL), then concentrated in vacuum. The residue was dissolved in Et0Ac
(2.00 L),
washed with aq.actric acid (500 mL x 2) and brine (500 mL), dried with
anhydrous Na2SO4,
filtered and concentrated in vacuum. The residue was washed with MTBE (1.00 L
x 2).
Afforded compound 13 (280 g, crude) as yellow solid.
[0375] 111 NMR: (400 MHz, DMSO-d6): 6 11.18 (s, 1H), 8.68 (s, 1H), 8.58 (s,
1H) 8.04 (d, J
= 7.2 Hz, 1H), 7.65-7.63 (m, 1H), 7.57-7.53 (m, 2H), 7.37-7.36 (m, 2H) 7.20-
7.28 (m, 10H),
7.07-7.09 (m, 2H), 6.85-6.82 (m, 6H), 6.07 (d, J= 4.0 Hz, 1H), 5.61 (d, J= 4.0
Hz, 1H), 5.24 (d,
J = 4.0 Hz, 1H), 4.79-4.78 (m, 1H), 4.34-4.32 (m, 1H), 4.12-4.11 (m, 1H), 3.74-
3.72 (m, 9H) ,
3.26-3.25 (m, 2H).
Example 21. General procedure for preparation of compound 14
N
)¨ aq. Na104
0
N N
DMTr0/46
Dioxane/H20, 25 C, 1 h DMTrOC3C
.--OH
0 0
13 14
[0376] To a solution of compound 13 (80.0 g, 119 mmol, 1.00 eq) in Dioxane
(1.20 L) was
added the solution of NaI04 (27.9 g, 130 mmol, 7.24 mL, 1.10 eq) in H20 (400
mL) at 25 C,
then stirred at 25 C for 1 h. TLC (Dichloromethane: Methanol = 15: 1, Rf=
0.32) showed no
starting material. The three batches were combined for working up and
purification. The
reaction mixture was filtered and the filtrate was used for next directly.
Afforded compound 14
(240 g, crude) as yellow oil. The crude was used for next directly.
Example 22. General procedure for preparation of compound 15
,--N ,--N
).¨NHBz N/1s
NaBH4
n Nr"-)N n In\
DMTr0¨ Dioxane/H20(5/2) , 25 C, 1 h DMTr0-
46A% yield
for 3 steps HO OH
14 15
[0377] To a solution of compound 14(80.0 g, 119 mmol, 1.00 eq) in dioxane
(1.20 L) was
added NaBH4 (4.96 g, 131 mmol, 1.10 eq) at 25 C, then stirred at 25 C for 1
h. TLC
(Dichloromethane: Methanol = 15: 1, Rf= 0.34) showed no starting material. The
reaction
mixture was added AcOH/Py (1/1, 50.0 mL), then concentrated in vacuum. The
three batches
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were combined with ET32454-7-P1A for working up and purification. The residue
was
extracted with Et0Ac (1.00 Lx 2), washed with aq.NaHCO3 (600 mL) and brine
(600 mL), dried
with anhydrous Na2SO4, filtered and concentrated in vacuum. The residue was
purified by
column chromatography (SiO2, DCM: Me0H = 50:1 to 10:1, 1% TEA). Afforded
compound 15
(230.0 g, 46.4% yield for 3 steps) as yellow solid.
[0378] LCMS: RT = 2.213 min, M-1 = 674.3.
[0379] 111 NMR: (400 MHz, DMSO-d6)
[0380] (511.25 (s, 1H), 8.75 (s, 1H), 8.67(s, 1H), 8.07-80.5 (m, 2H), 7.67-
7.63 (m, 1H), 7.57-
7.53 (m, 2H), 7.22-7.12 (m, 3H), 7.07-7.05 (m, 1H), 6.97-6.93 (m, 4H), 6.79-
6.77 (m, 4H), 6.05
(t, J= 12 Hz, 1H), 5.31-5.26 (m, 1H), 4.83 (t, J= 8.0 Hz, 1H), 4.14-4.04 (m,
2H), 3.84 (s, 1H),
3.69 (s, 6H), 3.49-3.47 (m, 2H), 2.89-2.78 (m, 1H), 2.77-2.75 (m, 1H).
Example 22. General procedure for preparation of compound 16
N// 14/\
BzCI, DBU
0 _____________________________ )11. 0 Nr---N
DMTrO DCM, -70 C, 1 h DMTrO
HO OH HO OBz
15 16
[0381] To a solution of compound 15 (145 g, 214 mmol, 1.00 eq) in DCM (2.00 L)
was added
DBU (65.3 g, 429 mmol, 64.7 mL, 2.00 eq), then cooled to -70 C and added BzCl
(33.2 g, 236
mmol, 27.4 mL, 1.10 eq) dropwise at -70 C, then stirred at -70 C for 1 h.
TLC
(Dichloromethane: Methanol = 10: 1, Rf= 0.43) showed no starting material. The
reaction
mixture was added Et0H (300 mL) at -70 C, then allowed to 25 C, washed with
aq.NaHCO3
(2.00 L x 2) and brine (1.00 L), dried over anhydrous Na2SO4, filtered and
concentrated in
vacuum. Afforded compound 16 (160 g, crude) as yellow solid. The crude was
used for next
directly.
Example 23. General procedure for preparation of compound 17
TBSCI, imidazole
Nr-i\N
DMTrO N.'"'N DCM, 25 C, 1 h DMTrO- N C.
HO OBz TBSO OBz
16 17
[0382] To a solution of compound 16 (160 g, 205 mmol, 1.00 eq) in DCM (960 mL)
was
added imidazole (21.0 g, 308 mmol, 1.50 eq) and TBSC1 (40.2 g, 267 mmol, 32.7
mL, 1.30 eq),
then stirred at 25 C for 1 h. TLC (Dichloromethane: Methanol = 10: 1, Rf=
0.51) showed no
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starting material. The reaction mixture was washed with aq.NaHCO3 (800 mL x 2)
and brine
(800 mL x 2), dried with anhydrous Na2SO4, filtered and concentrated in
vacuum. Afforded
compound 17 (160 g, crude) as yellow solid. The crude was used for next
directly.
Example 23. General procedure for preparation of compound 18
,..--N ,--N
Ni//--NHBz N/I\ )--NHBz
NaOH (1 M)
N
_______________________________ ).- o nN
DMTrO N''' Py:Me0H(2:1), DMTrO¨C.--''./
0 C, 0.5 h
TBSO OBz 21.7% yield TBSO OH
for 3 steps
17 18
To a solution of compound of 17 (200 g, 224 mmol, 1.00 eq) in Py (1.20 L) and
Me0H (480
mL) was added NaOH (1 M, 224 mL, 1.00 eq) dropwise at 0 C, then stirred at 0
C for 0.5 h.
TLC (Dichloromethane: Methanol = 10: 1, Rf= 0.42) showed no starting material.
The reaction
mixture was extracted with Et0Ac (1.00 Lx 2), washed with aq.NH4C1 (1.00 Lx 2)
and brine
(800 mL), dried with anhydrous Na2SO4, filtered and concentrated in vacuum.
ET32454-18-
PIA was combined with ET32454-19-P1A for purification. The residue was
purified by column
chromatography (SiO2, Petroleum ether/Ethyl acetate = 10/1 to DCM: Me0H =
20:1, 1% TEA).
Afforded compound 18 (58 g, 21.7% yield for 3 steps) as white solid.
[0383] 111 NMR: (400 MHz, DMSO-d6) 6 11.24 (s, 1H), 8.75 (s, 1H), 8.70 (s,
1H), 8.07-8.05
(m, 2H), 7.69-7.65 (m, 1H), 7.59-7.56 (m, 2H), 7.24-7.20 (m, 2H), 7.17-7.13
(m, 1H), 7.08-7.06
(m, 1H), 6.99-6.94 (m, 4H), 6.81-6.78 (m, 4H), 6.07-6.04 (m, 1H), 5.30-5.27(t,
J = 12 Hz, 1H),
4.20-4.14 (m, 1H), 4.06-3.99 (m, 1H), 3.88-3.85 (m, 1H), 3.76-3.70 (m, 7H),
3.61-3.57 (m, 1H),
0.80 (s, 9H), 0.03 (d, J= 12 Hz, 6H).
[0384] Example 24. General procedure for preparation of compound 19
,--N
//µ ,--NHI3z
N N
r \ --
NHBz Br
N a
NaH ,1 ) ).- )------NN
DMTr0 #N
DMTrOON,, DMF, 0-15 C, 16 h
TBSO 0
-11360 OH
H
0
18 19
To a solution of NaH (9.52 g, 238 mmol, 60% purity, 3.30 eq) in DIVIF (300 mL)
was added the
solution of compound 18 (57.0 g, 72.2 mmol, 1.00 eq) dropwise at 0 C, then
stirred at 0 C for
1 h. The mixture was added compound a (15.0 g, 108 mmol, 10.2 mL, 1.50 eq)
dropwise at 0
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C, and then stirred at 15 C for 15 h. TLC (Dichloromethane: Methanol = 10: 1,
Rf= 0.43)
showed no starting material. The reaction mixture was poured into aq.NH4C1
(2.00 L), extracted
with Et0Ac (800 mL x 2), washed with brine (500 mL x 2), dried with anhydrous
Na2SO4,
filtered and concentrated in vacuum. Afforded compound 19 (60.0 g, crude) as
yellow oil. The
crude was used for next directly.
[0385] LCMS: RT = 3.219 min, MS-1 = 846.4.
Example 25. General procedure for preparation of compound 20
iN.¨NHBz ,--N
\ ,.........Z. Nil \ NHBz
N.._ N
DMTrO0CN.,./I NI-14F, TEA
IN. DMTrO0C ¨
HO TBSO
Me0H, 15 C, 16 h 0 0
28.6% yield
for 2 steps
0 0
19 20
[0386] To a solution of compound 19 (60.0 g, 70.8 mmol, 1.00 eq) in Me0H (360
mL) was
added NH4F (21.0 g, 566 mmol, 8.00 eq) and TEA (14.3 g, 142 mmol, 19.7 mL,
2.00 eq) at 15
C, then stirred at 15 C for 16 h. TLC (Dichloromethane: Methanol = 10: 1, Rf=
0.33) showed
no starting material. The reaction mixture was concentrated in vacuum. The
residue was
purified by column chromatography (5i02, DCM: Me0H = 100:1 to 10:1, 1% TEA).
Afforded
compound 20 (16.5 g, 22.5 mmol, 28.6% yield) as white solid.
[0387] 111 NMR: (400 MHz, DMSO-d6) 6 11.24 (s, 1H), 8.76 (s, 1H), 8.71 (s,
1H), 8.07-8.05
(m, 2H), 7.69-7.65 (m, 1H), 7.59-7.56 (m, 2H), 7.24-7.20 (m, 2H), 7.17-7.13
(m, 1H), 7.08-7.06
(m, 1H), 6.99-6.94 (m, 4H), 6.81-6.78 (m, 4H), 6.25-6.22 (m, 1H), 4.84-4.82(t,
J = 6 Hz, 1H),
4.30-4.25 (m, 1H), 4.09-4.05 (m, 1H), 3.87-3.85 (m, 1H), 3.69 (d, J= 2 Hz,
7H), 3.62-3.60 (m,
2H), 3.50-3.47 (m, 2H), 3.39-3.37 (m, 2H), 3.17(s, 3H), 2.90-2.86 (m, 1H),
2.77-2.73 (m, 1H).
Example 26. General procedure for preparation of A-UNA-2'MOE 3'Phosphoramidite
)`rsib ,--N
,--N I Nll \ NHBz
Ny¨NHBz
N 0
DCI
DMTrO
DMTrO N-'11 v.
DCM, 0-15 C, 2 h e
HO 1 0
0
H34.2% yield NCc:(kN2\ H
0 0
20 A-UNA-2'MOE 3'Phosphoramidite
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[0388] To a solution of compound 20(15.0 g, 20.4 mmol, 1.00 eq) in dry DCM
(90.0 mL) was
added DCI (3.62 g, 30.7 mmol, 1.50 eq) and compound b (10.5 g, 34.8 mmol, 11.0
mL, 1.70 eq)
at 0 C, then stirred at 15 C for 2 h. TLC (Dichloromethane: Acetone = 3:1,
Rf= 0.53) showed
no starting material. The solution was poured into the silica gel column
directly. The reaction
mixture was purified by column chromatography (SiO2, Petroleum ether/Ethyl
acetate = 5/1 to
0/1, 1% TEA). Afforded A-UNA-2'MOE 3'Phosphoramidite (7.00 g, 34.2% yield) as
white
solid.
[0389] 111 NMR: (400 MHz, DMSO-d6) 6 11.26 (s, 1H), 8.77-8.72 (m, 2H), 8.06
(d, J= 7.6
Hz, 2H), 7.63 (t, J= 14.8 Hz, 1H), 7.56 (t, J= 15.2 Hz, 2H), 7.20-7.12 (m,
3H), 7.08-7.04 (m,
2H), 6.98-6.92 (m, 4H), 6.79-6.76 (m, 4H), 6.22 (t, J= 12 Hz, 1H), 4.27-4.24
(m, 1H), 4.04-3.98
(m, 2H), 3.69-3.66 (m, 10H), 3.63-3.62 (m, 2H), 3.62-3.60 (m, 2H), 3.39-3.37
(m, 2H), 3.17(s,
3H), 2.96-2.71 (m, 4H), 1.19-1.03 (m, 12H).
31P NMR: (162 MHz, DMSO-d6) 6 147.62, 147.46
HPLC: 97.8% purity
[0390] MS: M-1 = 932.2
Example 27. General procedure for preparation of compound 22
NH N11---NH
0 Ne¨NH
)
DMTrCI
0 DMTrO
N N Py, 0-15 C, 2 h
H0/4
H0µ..
HOs
21 22
[0391] To a solution of compound 21 (150.0 g, 424.5 mmol, 1.00 eq) in Py (900
mL) was
added DMTC1 (158.2 g, 467.0 mmol, 1.10 eq) at 0 C, then stirred at 15 C for 2
h. TLC
(Dichloromethane: Methanol = 20: 1, Rf= 0.38) showed no starting material. The
reaction
mixture was added Me0H (150 mL), then concentrated in vacuum. The residue was
dissolved
in Et0Ac (1000 mL), washed with aq.actric acid (500 mL x 2) and brine (500
mL), dried with
anhydrous Na2SO4, filtered and concentrated in vacuum. The residue was washed
with MTBE
(1000 mL x 2). Afforded compound 22 (284.0 g, crude) as yellow solid.
[0392] 111 NMR: (400 MHz, DMSO-d6) 6 12.10 (s, 1H), 11.66 (s, 1H), 8.58 (d, J=
4.0 Hz,
1H), 8.12 (s, 1H), 7.38-7.35 (m, 3H), 7.33-7.25 (m, 8H), 7.23-7.20 (m, 1H),
6.84-6.80 (m, 4H),
5.86 (d, J= 4.8 Hz, 1H), 5.62 (d, J= 5.6 Hz, 1H), 5.19 (d, J= 5.6 Hz, 1H),
4.54-4.53 (m, 1H),
4.22-4.20 (m, 1H), 3.74 - 3.72 (m, 7H), 3.25-3.18 (m, 1H), 2.78-2.76 (m, 1H),
2.76-2.75 (m,
1H), 1.13- 1.10 (m, 6H).
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Example 28. General procedure for preparation of compound 23
NH H
NNH
Na104.
DMTrOC)-
0 ----
N\N
Dioxane/H20, 15 C, 1 h DMTrO C"N
.-oH e
22 23
[0393] Three batches were carried in parallel. To a solution of compound 22
(80.0 g, 122.0
mmol, 1.00 eq) in dioxane (1600 mL) was added the solution of NaI04 (26.1 g,
122.0 mmol,
6.76 mL, 1.00 eq) in H20 (640 mL) at 15 C, then stirred at 15 C for 1 h. TLC
(Dichloromethane: Methanol = 15: 1, Rf= 0.28) showed no starting material. The
reaction
mixture was filtered and the filtrate was used for next directly. Afforded
compound 23 (239.3 g,
crude) as yellow oil. The crude was used for next directly.
Example 29. General procedure for preparation of compound 24
H
0 .T;Ir0 0 rrO
NaBH4
Dioxane, 15 C, 1 h
mr)N
DMTrO N-'" 66.3% yield
for 3 steps
HO OH
23 24
[0394] To a solution of compound 23 (79.8 g, 122.0 mmol, 1.00 eq) in dioxane
(1400 mL)
was added NaBH4 (4.62 g, 122.0 mmol, 1.00 eq) at 15 C, then stirred at 15 C
for 1 h. TLC
(Dichloromethane: Methanol = 15: 1, Rf= 0.29) showed no starting material. The
reaction
mixture was added AcOH/Py (1/1, 40 mL), then concentrated in vacuum. The three
batches
were combined for working up and purification. The residue was extracted with
Et0Ac (1000
mL x 2), washed with aq.NaHCO3 (600 mL) and brine (600 mL), dried with
anhydrous Na2SO4,
filtered and concentrated in vacuum. The residue was purified by column
chromatography
(SiO2, DCM: Me0H = 50:1 to 10:1, 1% TEA). Afforded compound 24 (206.0 g, 66.3%
yield
for 3 steps) as yellow solid.
[0395] 11-1 NMR: (400 MHz, DMSO-d6) 6 12.11 (s, 1H), 11.71 (s, 1H), 8.17 (s,
1H), 7.21-7.19
(m, 2H), 7.10-7.01 (m, 3H), 6.98-6.96 (m, 4H), 6.80-6.77 (m, 4H), 5.71 (t, J =
6.4 Hz, 1H), 5.21
(t, J = 5.6 Hz, 1H), 4.78 (t, J = 5.2 Hz, 1H), 3.98-3.97 (m, 2H), 3.97-3.76
(m, 1H), 3.71 (s, 6H),
3.44-3.42 (m, 2H), 2.86-2.75 (m, 3H), 1.12-1.06 (m, 6H).
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Example 30. General procedure for preparation of compound 25
JH
II 0 )/: O 0 II
DBU, BzCI
DCM, -78 C, 1 h r\N1
HO HO OH OBz
24 25
[0396] To a solution of compound 24 (93.0 g, 141.4 mmol, 1.00 eq) in DCM (1860
mL) was
added DBU (43.1 g, 282.8 mmol, 42.6 mL, 2.00 eq), then cooled to -78 C and
added BzCl
(21.9 g, 155.5 mmol, 18.1 mL, 1.10 eq) dropwise at -78 C, then stirred at -78
C for 1 h. TLC
(Dichloromethane: Methanol = 15: 1, Rf= 0.38) showed no starting material. The
reaction
mixture was added Et0H (200 mL) at -78 C, then allowed to 25 C, washed with
aq.NaHCO3
(600 mL x 2) and brine (600 mL), dried over anhydrous Na2SO4, filtered and
concentrated in
vacuo. Afforded compound 25 (224.0 g, crude) as yellow solid. The crude was
used for next
directly.
Example 31. General procedure for preparation of compound 26
H
If 0 r rO 0 r If
rO
TBSCI, imidazole
DCM, 15 C, 2 h
r\N
0
DMTrO DMTre.4jr
HO OBz TBSO OBz
25 26
[0397] To a solution of compound 25 (240.0 g, 315.0 mmol, 1.00 eq) in DCM
(1440 mL) was
added imidazole (32.2 g, 472.6 mmol, 1.50 eq) and TBSC1 (61.7 g, 409.6 mmol,
50.2 mL, 1.30
eq), then stirred at 15 C for 2 h. TLC (Dichloromethane: Methanol = 20: 1,
Rf= 0.38) showed
no starting material. The reaction mixture was washed with aq.NaHCO3 (400 mL x
2) and brine
(400 mL x 2), dried with anhydrous Na2SO4, filtered and concentrated in
vacuum. Afforded
compound 26 (263.0 g, crude) as yellow solid. The crude was used for next
directly.
Example 31. General procedure for preparation of compound 27
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H HH
0 0 0
NaOH (1 M)
Me0H/Py
DMTrOAYN 0 C, 15 min DMTrOIDYNN
TBSO OBz TBSO ()H
26 27
[0398] To a solution of compound of 26 (240.0 g, 274 mmol, 1.00 eq) in Py
(1200 mL) and
Me0H (480 mL) was added NaOH (1 M, 313 mL, 1.14 eq) dropwise at 0 C, then
stirred at 0 C
for 15 min. TLC (Dichloromethane: Methanol = 20: 1, Rf= 0.21) showed no
starting material.
The reaction mixture was extracted with Et0Ac (1000 mL x 2), washed with
aq.NH4C1 (1000
mL x 2) and brine (1000 mL), dried with anhydrous Na2SO4, filtered and
concentrated in
vacuum. The residue was purified by column chromatography (SiO2, Petroleum
ether/Ethyl
acetate = 10/1 to DCM: Me0H = 20:1, 1% TEA). Afforded compound 27 (50 g, 21.6%
yield)
as white solid.
[0399] 11-1 NMR: (400 MHz, DMSO-d6) 6 12.09 (br s, 1H), 11.64 (br s, 1H), 8.20
(s, 1H),
7.21-7.10 (m, 2H), 7.10-7.01 (m, 3H), 6.99-6.95 (m, 4H), 6.80-6.76 (m, 4H),
5.64 (t, J= 6.4 Hz,
1H), 5.16 (t, J= 6.0 Hz, 1H), 4.07-4.05 (m, 1H), 3.93-3.92 (m, 1H), 3.71-3.51
(m, 8H), 2.85-
2.82 (m, 1H), 2.80-2.50 (m, 3H), 1.10 (t, J= 6.0 Hz, 6H), 0.74 (s, 9H), -0.06
(d, J = 13.6 Hz,
6H).
Example 32. General procedure for preparation of compound 28
Na
0
DMF, 0511 C, 16 h DMTrO"
TBSO
TBSe OH
27 28
[0400] To a solution of NaH (7.52 g, 188.1 mmol, 60% purity, 3.30 eq) in DIVIF
(132 mL) was
added the solution of compound 27 (44.0 g, 57.0 mmol, 1.00 eq) in DIVIF (132
mL) dropwise at
0 C, then stirred at 0 C for 1 h. The mixture was added compound a (11.9 g,
85.5 mmol, 8.03
mL, 1.50 eq) dropwise at 0 C, and then stirred at 15 C for 15 h. TLC
(Dichloromethane:
Methanol = 10: 1, Rf= 0.34) showed no starting material. The reaction mixture
was poured into
aq.NH4C1 (1500 mL), extracted with Et0Ac (500 mL x 2), washed with brine (500
mL), dried
with anhydrous Na2SO4, filtered and concentrated in vacuum. Afforded compound
28 (47.3 g,
crude) as yellow oil. The crude was used for next directly.
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Example 33. General procedure for preparation of compound 29
H H
rro rro
NI-14F, TEA nN
Me0H, 15-40 C, 6 h INATreST-,C.
38.0% yield
TBSO 0 HO 0
for 2 steps
0 0
28 29
[0401] To a solution of compound 28 (47.3 g, 57.0 mmol, 1.00 eq) in Me0H (280
mL) was
added NH4F (16.9 g, 456.0 mmol, 8.00 eq) and TEA (8.00 g, 79.1 mmol, 11.00 mL,
1.39 eq) at
15 C, then stirred at 40 C for 6 h. TLC (Dichloromethane: Methanol = 10: 1,
Rf= 0.33)
showed no starting material. The reaction mixture was concentrated in vacuum.
The residue
was purified by column chromatography (SiO2, DCM: Me0H = 100:1 to 10:1, 1%
TEA).
Afforded compound 29 (15.5 g, 21.6 mmol, 38.0% yield) as white solid.
[0402] 111 NMR: (400 MHz, DMSO-d6) 6 12.11 (s, 1H), 11.73 (s, 1H), 8.24 - 8.23
(m, 1H),
7.22 - 7.01 (m, 5H), 6.99 ¨ 6.96 (m, 4H), 6.80 - 6.77 (m, 4H), 5.91 - 5.85 (m,
1H), 4.79 (t, J=
2.6 Hz, 1H), 4.13 -4.04 (m, 1H), 4.02 ¨ 4.00 (m, 2H), 3.73-3.71 (m, 7H), 3.57-
3.56 (m, 2H),
3.45-3.43 (m, 2H), 3.38-3.37 (m, 2H), 3.17 (s, 3H), 2.81-2.77 (m, 3H), 1.14-
1.10 (m, 6H).
Example 34. General procedure for preparation of G-UNA-2'MOE 3'Phosphoramidite
H ,J. J,
N H
0 N" N'
0 );_ir
N
DCI
DMTrO yNN
N
DCM, 0-15 C, 2 h DMTrOyN
He KO 31.4% yield
? I ?,1
0 0
29 G-UNA-2'MOE 3'Phosphoramidite
[0403] To a solution of compound 29 (15.0 g, 21.0 mmol, 1.00 eq) in DCM (90
mL) was
added DCI (3.71 g, 31.4 mmol, 1.50 eq) and compound b (10.7 g, 35.6 mmol,
11.31 mL, 1.70
eq) at 0 C, then stirred at 15 C for 2 h. TLC (Dichloromethane: Acetone =
2:1, Rf= 0.53)
showed no starting material. The solution was poured into the silica gel
column directly. The
reaction mixture was purified by column chromatography (SiO2, Petroleum
ether/Ethyl acetate =
5/1 to 0/1, 1% TEA). ET32432-31-PlA was combined with ET32432-34-P1A for
analysis and
delivery. Afforded G-UNA-2'MOE 3'Phosphoramidite (6.5 g, 31.4% yield) as
yellow solid.
[0404] 111 NMR: (400 MHz, DMSO-d6) 6 12.09 (s, 1H), 11.66 (s, 1H), 8.22 (s,
1H), 7.21-6.99
(m, 5H), 6.99-6.98 (m, 4H), 6.80-6.76 (m, 4H), 5.83 (t, J= 6.0 Hz, 1H), 4.16-
4.15 (m, 1H),
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3.89-3.71 (m, 1H), 3.66-3.65 (m, 1H), 3.58-3.56 (m, 12H), 3.39-3.38 (m, 2H),
3.37-3.17 (m,
2H), 3.17 (s, 3H), 2.82-2.72 (m, 3H), 2.72-2.70 (m, 2H), 1.12-1.09 (m, 12H),
1.02-0.99 (m, 6H).
[0405] 31PNMR: (162 MHz, DMSO-d6) 6 147.46
[0406] HPLC: 95.8% purity
[0407] MS: M-1 = 914.2
[0408] Example 35. Exemplary MSTN siRNA guide strands and passenger strands
comprising UNASense strand and antisense strand sequences targeting MSTN mRNA
were
generated to include one or more UNAs and/or one or more modified
internucleotide linkages.
siRNAs comprising the UNA-modified sense strand and/or antisense strands were
tested for its
stability (Tm) and the efficacy of the siRNA in two different doses (lnIVI and
0.1 nM,
respectively). The sequences and test results are shown in Tables 5 and 6.
TABLE 5
SEQ GS (guide, antisense) SEQ PS (passenger, sense)
Tm 1 nM 0.1 nM
ID ID (% (%
NO NO
mRNA mRNA)
1423 vpUqsUfsauuAfuuuguucUfuUfgc 142 gsgscaaaGfAfAfcaaauaausa 64.57 21.8 50.4
9 csusu 40 sa
1424 vpUqsUfsauuAfuuuguucUfuUfgc 142 GqGqcaaaGfAfAfcaaauaau 55.4 17.3 37.3
1 csusu 42 AqAq
1424 vpUqsUfsauuAfuuuguucUfuUfgc 142 GqsGqcaaaGfAfAfcaaauaau 54.56 19.9 45.9
3 csusu 44 AqsAq
1424 vpUqsUfsauuAfuuuguucUfuUfgc 142 gsgscaaaGfAfAfcaaauaauA 66.05 20.6 45.6
csusu 46 qAq
1424 vpUqsUfsauuAfuuuguucUfuUfgc 142 GqGqcaaaGfAfAfcaaauaaus 57.64 21.6 43.1
7 csusu 48 asa
1424 vpUqsUfsauuAfuuuguucUfuUfgc 142 GqGqCqaaaGfAfAfcaaauaa 46.72 21.5 52.4
9 csusu 50 Uqaqaq
1425 vpUqsUfsauuAfuuuguucUfuUfgc 142 GqGqCqAbaaGfAfAfcaaaua 51.31 18.7 39.5
1 csusu 52 AbUqAqAq
1425 vpUqsUfsauuAfuuuguucUfuUfgc 142 GbGbcaaaGfAfAfcaaauaau 74.06 16.1 35.6
3 csusu 54 AqAq
1425 vpUqsUfsauuAfuuuguucUfuUfgc 142 GqGqcaaaGfAfAfcaaauaau 62.59 15.8 34.9
5 csusu 56 AbAb
vp: vinylphosphate
s: phosphorothioate internucleotide linkage
Xf = 2'-fluoro nucleoside
lower case: 2'-0-methyl
Xb: LNA
TABLE 6
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SEQ GS (guide, antisense) SEQ PS (passenger, sense) Tm
1 nM 0.1 nM
ID ID (%
(%
NO NO
mRNA) mRNA)
1425 vpUqsUfsauuAfuuuguucUfuUfgc 142 gsgscaaaGfAfAfcaaauaausa 66.4 24.6 45.4
7 csusu 58 sa
1425 vpUqsUfsauuAfuuuguucUfuUfgc 142 gsgscaaaGfAfAfcaaauaausa 66.6 20.6 40.6
9 cUqUq 60 sa
1426 vpUqsUfsauuAfuuuguucUfuUfG 142 gsgscaaaGfAfAfcaaauaausa 51.5 37.3 72.1
1 qCqCqsusu 62 sa
1426 vpUqsUfsauuAfuuuguucUfuUfG 142 gsgscaaaGfAfAfcaaauaausa 51.5 29.4 61.5
3 qCqCqTbTb 64 sa
1426 vpUqsUfsauuAfuuuguucUfuUfG 142 gsgscaaaGfAfAfcaaauaausa 68.2 49.7 83.7
bCbCbUqUq 66 sa 6
1426 vpUqsUfsauuAfuuuguucUfuUfG 142 gsgscaaaGfAfAfcaaauaausa 52.6 31.7 68.9
7 qCqCqUqUq 68 sa
1426 vpUqsUfsauuAfuuuguucUfuUfgc 142 gsgscaaaGfAfAfcaaauaausa 66.2 16.0 34.8
9 cUqsUq 70 sa 5
1427 vpUqsUfsauuAfuuuguucUfuUfgc 142 gsgscaaaGfAfAfcaaauaausa 66.3 18.9 41.3
1 csUqsUq 72 sa 5
1427 vpUqsUfsauuAfuuuguucUfuUfgc 142 gsgscaaaGfAfAfcaaauaausa 64.5 24.9 47.8
3 csUwsUw 74 sa
1427 vpUqsUfsauuAfuuuguucUfuUfgc 142 gsgscaaaGfAfAfcaaauaausa 65.4 21.9 43.7
5 csUxsUx 76 sa
1427 vpUqsUfsauuAfuuuguucUfuUfgc 142 gsgscaaaGfAfAfcaaauaausa 64.9 22.5 37.5
7 csUysUy 78 sa 7
1427 vpUqsUfsauuAfuuuguucUfuUfgc 142 gsgscaaaGfAfAfcaaauaausa 66.5 21.0 36.6
9 cUwUw 80 sa 5
1428 vpUqsUfsauuAfuuuguucUfuUfgc 142 gsgscaaaGfAfAfcaaauaausa 64.8 15.9 36.0
1 cUxUx 82 sa
1428 vpUqsUfsauuAfuuuguucUfuUfgc 142 gsgscaaaGfAfAfcaaauaausa 66.3 18.8 38.1
3 cUyUy 84 sa 7
1428 vpUqsTbauuAfuuuguucUfuUfgcc 142 gsgscaaaGfAfAfcaaauaausa 68.3 10.7 34.0
5 UqsUq 86 sa 6
1428 vpUqsTbauuAfuuuguucUfuUfgcc 142 gsgscaaaGfAfAfcaaauaausa 66.6 14.0 37.2
7 UwsUw 88 sa 6
1428 vpUqsTbauuAfuuuguucUfuUfgcc 142 gsgscaaaGfAfAfcaaauaausa 67.8 13.5 34.0
9 UxsUx 90 sa 5
1429 vpUqsTbauuAfuuuguucUfuUfgcc 142 gsgscaaaGfAfAfcaaauaausa 68.4 20.6 43.7
1 UysUy 92 sa 6
1429 vpUqsUqsauuAfuuuguucUfuUfg 142 gsgscaaaGfAfAfcaaauaausa 65.9 89.8 103.6
3 ccsusu 94 sa 2
1429 vpUqsUfsauuAquuuguucUfuUfg 142 gsgscaaaGfAfAfcaaauaausa 58.3 63.1 88.8
5 ccsusu 96 sa 4
1429 vpUqsUfsauuAfuuuguucUquUfg 142 gsgscaaaGfAfAfcaaauaausa 57.2 47.4 88.4
7 ccsusu 98 sa 6
1429 vpUqsUfsauuAfuuuguucUfuUqg 143 gsgscaaaGfAfAfcaaauaausa 57.4 19.5 48.2
9 ccsusu 00 sa 5
1430 vpUqsUwsauuAfuuuguucUfuUfg 143 gsgscaaaGfAfAfcaaauaausa 65.7 93.3 85.7
1 ccsusu 02 sa
1430 vpUqsUfsauuAfuuuguucUwuUfg 143 gsgscaaaGfAfAfcaaauaausa
101.6 106.5
3 ccsusu 04 sa
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1430 vpUqsUfsauuAfuuuguucUfuUwg 143 gsgscaaaGfAfAfcaaauaausa 57.4 24.2 48.4
ccsusu 06 sa
1430 vpUqsUxsauuAfuuuguucUfuUfgc 143 gsgscaaaGfAfAfcaaauaausa 69.7 103.4 113.4
7 csusu 08 sa
1430 vpUqsUfsauuAfuuuguucUxuUfgc 143 gsgscaaaGfAfAfcaaauaausa 55.3 60.2
83.3
9 csusu 10 sa 1
1431 vpUqsUfsauuAfuuuguucUfuUxgc 143 gsgscaaaGfAfAfcaaauaausa 56.8 21.9
47.1
1 csusu 12 sa 5
1431 vpUqsUysauuAfuuuguucUfuUfgc 143 gsgscaaaGfAfAfcaaauaausa 68.7 94.8
104.2
3 csusu 14 sa
1431 vpUqsUfsauuAfuuuguucUyuUfgc 143 gsgscaaaGfAfAfcaaauaausa 56.5 66.3
92.8
5 csusu 16 sa
1431 vpUqsUfsauuAfuuuguucUfuUygc 143 gsgscaaaGfAfAfcaaauaausa 57.3 20.7
46.0
7 csusu 18 sa 9
1431 vpUqsTbsAquuAfuuuguucUfuUf 143 gsgscaaaGfAfAfcaaauaausa 61.6 69.8 94.0
9 gccsusu 20 sa
1432 vpUqsUfsauuAbUquuguucUfuUf 143 gsgscaaaGfAfAfcaaauaausa 61.7 64.6 93.9
1 gccsusu 22 sa 4
1432 vpUqsUfsauuAfUquuguucUfuUf 143 gsgscaaaGfAfAfcaaauaausa 51 56.2 82.6
3 gccsusu 24 sa
1432 vpUqsUfsauuAfuuuguucUfuTbG 143 gsgscaaaGfAfAfcaaauaausa 59.4 24.9 57.5
5 qccsusu 26 sa 2
1432 vpUqsUfsauuAfuuuguucUfUqTb 143 gsgscaaaGfAfAfcaaauaausa 50 80.5 93.5
7 Gqccsusu 28 sa
1432 vpUqsTbsAqUqUqAbuuuguucUf 143 gsgscaaaGfAfAfcaaauaausa 66.5 108.2 98.6
9 uUfgccsusu 30 sa
1433 vpUqsTbsAquUqAbUquuguucUf 143 gsgscaaaGfAfAfcaaauaausa 58.9 102.0 102.8
1 uUfgccsusu 32 sa 9
1433 vpUqsTbsauuAbUquuguucUfuUf 143 gsgscaaaGfAfAfcaaauaausa 62.1 83.2 95.7
3 gccsusu 34 sa 6
1433 vpUqsUfsauuAfuuuguucTbuTbG 143 gsgscaaaGfAfAfcaaauaausa 55.1 54.1 77.4
5 qCqCqsusu 36 sa 7
1433 vpUqsUfsauuAfuuuguuCqTbUqT 143 gsgscaaaGfAfAfcaaauaausa 37 82.2 97.0
7 bGqccsusu 38 sa
1433 vpUqsUfsauuAquuuguucUfuUqg 143 gsgscaaaGfAfAfcaaauaausa 44.7 74.8 99.9
9 ccsusu 40 sa
1434 vpUqsTbsauuAquuuguucTbuUqg 143 gsgscaaaGfAfAfcaaauaausa 49.9 89.8 89.3
1 ccsusu 42 sa
1434 vpUqsTbauuAquuuguucTbuUqg 143 gsgscaaaGfAfAfcaaauaausa 52.8 90.6 96.1
3 ccusu 44 sa
vp: vinylphosphate
s: phosphorothioate internucleotide linkage
Xf = 2'-fluoro nucleoside
lower case: 2'-0-methyl
Xb: LNA
Example 36. MSTN siRNA guide strands and passenger strands comprising UNA and
their efficacies in downregulating MSTN mRNA in vitro
[0409] Four candidate siRNAs targeting MSTN mRNA were tested for its efficacy
to
downregulate MSTN mRNA in vitro in SJCRH30 cells. The sequences of four
candidate
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siRNAs (R002728-1, R002729-1, R002734-1, R002735-1) and their stability
(indicated by Tm)
and efficacy (EC50 value) are shown in Table 7. FIG. 3 also shows
downregulation of MSTN
mRNA in SICRH30 cells by each candidate siRNA.
TABLE 7
siRNA SEQ ID PS (passenger, sense) SEQ ID GS (guide,
antisense) EC50
NO NO (PM) Tm
[C]
aminoC6-
vpUqsUfsauuAfuuug
R002728-1 14345 gsgscaaaGfAfAfcaaauaa 14346 18.41
uucUfuUfgccUqUq
67.51
usasa
aminoC6-
vpUqsUfsauuAfuuug
R002729-1 14347 gsgscaaaGfAfAfcaaauaa 14348 25.27
uucUfuUfgccUqsUq
67.5
usasa
aminoC6-
vpUqsUfsauuAfuuug
R002734-1 14349 GqGqcaaaGfAfAfcaaaua 14350 39.45
uucUfuUfgccsusu 54
auAqAq
aminoC6-
vpUqsUfsauuAfuuug
R002735-1 14351 GqsGqcaaaGfAfAfcaaau 14352 45.19
uucUfuUfgccsusu
54.8
aauAqsAq
vp: vinylphosphate
s: phosphorothioate internucleotide linkage
Xf = 2'-fluoro nucleoside
lower case: 2'-0-methyl
Xb: LNA
Example 37. MSTN siRNA guide strands and passenger strands comprising UNA and
their efficacy in downregulating MSTN mRNA in vivo
[0410] Four siRNAs targeting MSTN mRNA were tested for its efficacy to
downregulate
MSTN mRNA in vivo. The sequences of four candidate siRNAs (R002728-1, R002729-
1,
R002734-1, R002735-1) and their stability (indicated by Tm) and efficacy (EC50
value) are
shown in Table 7. siRNA targeting MSTN mRNA was conjugated with a-CD71
antibody, and
the a-CD71-MSTN siRNA were administered in vivo to determine the effect of the
a-CD71-
MSTN siRNA. Table 8 shows Kd (% mRNA) value and the concentration of siRNAs in
the
Gastronecmius muscle after administering the siRNA conjugates. FIG. 4 shows a
comparison
graph of the four MSTN siRNA conjugates.
TABLE 8
siRNA KD in Gastroc siRNA in
Gastroc
[% mRNA] [nM]
d14 d28 d44 d57 d14 d28 d44
d57
R002728-1 27 54 75 92
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R002729-1 16 22 51 52 19.8 8.0 3.4 2.2
R002734-1 33 44 59 59 8.0 2.4 0.9 0.3
R002735-1 24 18 18 28 14.0 5.1 2.4 0.6
[0411] While preferred embodiments of the present disclosure have been shown
and described
herein, it will be obvious to those skilled in the art that such embodiments
are provided by way
of example only. Numerous variations, changes, and substitutions will now
occur to those
skilled in the art without departing from the disclosure. It should be
understood that various
alternatives to the embodiments of the disclosure described herein may be
employed in
practicing the disclosure. It is intended that the following claims define the
scope of the
disclosure and that methods and structures within the scope of these claims
and their equivalents
be covered thereby.
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Representative Drawing